A complex that inhibits viral RNA replication and transcription

Abstract

To provide complexes that target RNA molecules having high binding strength and selectivity to RNA higher order structures of RNA molecules, and to provide transcription inhibitors of viral RNA, viral RNA replication inhibitors, pharmaceutical compositions, methods of inhibiting the transcription or replication of viral RNA, screening methods for compounds having viral RNA replication- or transcription-inhibiting ability, and kits for evaluating the RNA replication- or transcription-inhibiting ability of a compound.SOLUTION: Provided is an RNA-targeting complex that extends from the 5'end to the 3'end and comprises: a first nucleic acid sequence; a second nucleic acid sequence located at the 3'-terminal side from the first nucleic acid sequence and forming a double strand with the first nucleic acid sequence; and an internal loop formed between the double strands. The complex comprises: a PNA chain that complementarily binds to a target nucleic acid sequence from the 5'end to the 8th or 9th base of the first nucleic acid sequence of RNA and has the same strand length as the target nucleic acid sequence; and 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolineamine (DPQ), and a linker that links the PNA chain to the DPQ.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to an RNA-targeting complex, a viral RNA transcription inhibitor, a viral RNA replication inhibitor, a pharmaceutical composition, a method for inhibiting viral RNA transcription or replication, a method for screening for a compound capable of inhibiting viral RNA replication or transcription, and a kit for evaluating the ability to inhibit viral RNA replication or transcription. [Background technology]

[0002] Influenza viruses are RNA viruses that infect humans and other animals and cause influenza, and have been causing great damage to humans and livestock for a long time.

[0003] Influenza viruses are divided into four types, A to D, but humans are infected with types A, B, and C. Influenza viruses A to C are a type of Orthomyxoviridae family, and are single-stranded RNA viruses with a membranous structure called an envelope. In particular, influenza viruses A and B are currently showing strong pathology and causing global pandemics. Symptoms of these two viruses typically include fever, headache, general malaise, muscle and joint pain, etc., which appear after an incubation period of about 1 to 3 days after infection, followed by upper respiratory tract symptoms such as cough and runny nose, and then improve within about a week. One of the characteristics of influenza viruses is that they replicate their genomes using viral RNA polymerase, and because of their high rate of mutation introduction, mutant strains can be easily isolated. Furthermore, because influenza viruses have segmented genomes, when different subtypes of the virus infect a cell at the same time, the viral genomes mix within the cell, and mutations occur that produce new viruses with genome combinations different from those of the original virus. Influenza A viruses are zoonotic, and there are more than 100 subtypes based on antigenic combinations. On the other hand, type B viruses are prevalent only in certain animals, including humans, and there are almost no subtypes. Therefore, only type A influenza carries the risk of new influenza viruses emerging, and among influenza viruses, type A viruses are the greatest threat.

[0004] Meanwhile, influenza treatment drugs that have been developed and approved in Japan so far can be roughly divided into four categories: "M2 protein inhibitors," "neuraminidase (NA) inhibitors," "RNA polymerase inhibitors," and "cap-dependent endonuclease inhibitors." All of these drugs are small molecules that inhibit specific processes in the influenza virus life cycle.

[0005] "M2 protein inhibitors" are drugs that inhibit the proton channel activity of M2 by incorporating one molecule into the M2 protein tetramer, thereby inhibiting the uncoating of virus particles. M2 protein inhibitors include amantadine (Symmetrel (registered trademark)) and rimantadine (Flumazin (registered trademark), but these "M2 protein inhibitors" are not currently used for treatment.

[0006] "Neuraminidase (NA) inhibitors" have a structure very similar to the host's sialic acid, and they enter the enzyme active site of NA and occupy the active site in place of sialic acid, thereby inhibiting the action of NA. Examples of NA inhibitors include oseltamivir (Tamiflu (registered trademark)), peramivir (Rapiacta (registered trademark)), zanamivir (Relenza (registered trademark)), and laninamivir (Inavir (registered trademark)).

[0007] "RNA polymerase inhibitors" act as nucleic acid base mimics and are incorporated during RNA synthesis, synthesizing RNA with a sequence different from the vRNA or cRNA that should be synthesized. This inhibits RNA replication and suppresses viral proliferation. These drugs are non-specific and have severe side effects, so they are rarely used except in emergency situations. Examples of RNA polymerase inhibitors include ribavirin (Copegus (registered trademark)) and favipiravir (Avigan (registered trademark)).

[0008] "Cap-dependent endonuclease inhibitors" bind to the active center of polymerase PA, which has nuclease activity, and thus cannot cleave the cap structure of the host cell's mRNA precursor, inhibiting the synthesis of viral mRNA. An example of a cap-dependent endonuclease inhibitor is baloxavir marboxil (Xofluza (registered trademark)). However, because this drug targets viral proteins, it has been pointed out that, like "M2 protein inhibitors" and "NA inhibitors," resistant strains with reduced binding ability due to mutations in specific amino acids may emerge.

[0009] In addition to existing denucleation inhibitors, release inhibitors, RNA replication inhibitors, and transcription inhibitors, individual laboratories are also developing molecules that target and inhibit the virus' life cycle, which are different from previous drugs, and in recent years, there has been active development of siRNA-based treatments and antibody drugs. However, most of the drugs developed still target proteins, and it has been reported that even when proteins are targeted, viruses can acquire drug resistance due to amino acid mutations.

[0010] The development of drugs that target highly conserved regions of viral RNA would be useful for preventing or treating viral infections.

[0011] The higher-order structure of RNA plays an important role in many biological phenomena. Intracellular RNA is known to have a complex higher-order structure that includes non-Watson-Crick base pairing sites such as loops, dangling ends, and internal loops in addition to the double-stranded structure (stem) formed by Watson-Crick hydrogen bonds.

[0012] The present inventors have previously succeeded in developing a fluorescent probe (tFIT probe) consisting of PNA with a labeling molecule attached, which forms a triplex structure with an RNA duplex structure in a sequence-selective manner under acidic conditions (Non-Patent Document 1), and have proposed a method for analyzing RNA duplex base sequences using this probe.

[0013] Although the development of molecules targeting RNA higher-order structures has been actively pursued, clear molecular design guidelines for expressing excellent binding strength and binding selectivity to the target have yet to be obtained. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] J. Am. Chem. Soc., 2016, 138, 9397-9400. Summary of the Invention [Problem to be solved by the invention]

[0015] The problem to be solved by the present invention is to provide an RNA-targeting complex having high binding affinity and selectivity for the RNA higher-order structure of RNA, a viral RNA transcription inhibitor, a viral RNA replication inhibitor, a pharmaceutical composition, a method for inhibiting viral RNA transcription or replication, a method for screening for a compound capable of inhibiting viral RNA replication or transcription, and a kit for evaluating the ability to inhibit viral RNA replication or transcription. [Means for solving the problem]

[0016] In order to achieve the above-mentioned object, the present inventors targeted the promoter region of influenza A virus RNA, which is particularly highly conserved, and discovered that a complex combining a PNA that binds sequence-selectively to the double-stranded structure of the promoter region and DPQ (6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) that binds to the internal loop of the promoter region unexpectedly and significantly inhibits RNA transcription and replication, thereby completing the present invention.

[0017] The present invention includes, for example, the embodiments described below.

[0018] Item 1. A complex targeting an RNA, comprising a first nucleic acid sequence extending from the 5' end to the 3' end, a second nucleic acid sequence located on the 3' end side of the first nucleic acid sequence and forming a double strand with the first nucleic acid sequence, and an internal loop formed between the double strand, a PNA strand that binds complementarily to a target nucleic acid sequence from the 5' end of the first nucleic acid sequence of the RNA to the 8th or 9th base and has the same strand length as the target nucleic acid sequence; 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ), a linker connecting the PNA strand and the DPQ; A complex comprising:

[0019] Item 2. The conjugate according to item 1, wherein at least one PNA constituting the PNA strand is substituted with methinecyanine.

[0020] Item 3. The complex according to Item 2, wherein in a double strand formed by the first nucleic acid sequence and the second nucleic acid sequence, the PNA on the PNA strand at a position complementary to the RNA on the first nucleic acid sequence where a base pair mismatch occurs is substituted with methinecyanine.

[0021] Item 4. The conjugate according to Item 2 or 3, wherein the methine cyanine is selected from thiazole orange (TO), quinoline blue (QB), and thiazole red (TR).

[0022] Item 5. The base sequence of the PNA strand is N-terminus-NTCNNTCTTT-C-terminus (wherein the first N is no base or C, and the fourth N is G, C, A, T or methinecyanine). Item 5. The complex according to any one of Items 1 to 4,

[0023] Item 6. The complex according to Item 1, wherein the RNA is influenza A virus RNA.

[0024] Item 7. A transcription inhibitor of viral RNA, comprising the complex according to any one of items 1 to 6.

[0025] Item 8. A viral RNA replication inhibitor comprising the complex according to any one of items 1 to 6.

[0026] Item 9. A pharmaceutical composition for inhibiting transcription or replication of viral RNA, comprising the complex according to any one of items 1 to 6.

[0027] Item 10. A pharmaceutical composition for preventing or treating a viral infection, comprising the complex according to any one of items 1 to 6.

[0028] Item 11. A method for inhibiting RNA transcription or replication in vitro or in a non-human mammalian subject, comprising: A method comprising contacting RNA with the complex according to any one of items 1 to 6, The RNA extends from the 5' end to the 3' end and comprises a first nucleic acid sequence, a second nucleic acid sequence located on the 3' end side of the first nucleic acid sequence and forming a double strand with the first nucleic acid sequence, and an internal loop formed between the double strand; The complex is a PNA strand that binds complementarily to a target nucleic acid sequence from the 5' end of the first nucleic acid sequence of the RNA to the 8th or 9th base and has the same strand length as the target nucleic acid sequence; 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ), a linker connecting the PNA strand and the DPQ; A method comprising:

[0029] Item 12. A method for screening a compound capable of inhibiting RNA replication or transcription, comprising: A step of previously binding RNA to the complex according to any one of items 1 to 6 to form a conjugate; contacting the conjugate with a test compound; and measuring each signal value obtained from the bound body before and after the contact; A screening method comprising:

[0030] Item 13. The method according to Item 12, wherein the test compound is determined to be a hit compound when the signal value after contact with the test compound is lower than the signal value before contact.

[0031] Item 14. A kit for evaluating the RNA replication or transcription inhibitory ability of a test compound, comprising: A kit comprising the complex according to any one of items 1 to 6.

[0032] Item 15. A kit for evaluating a viral infection, comprising: A kit comprising the complex according to any one of items 1 to 6.

[0033] Item 16. An in vitro test for evaluating a viral infection, comprising: Item 7. An in vitro test agent comprising the complex according to any one of Items 1 to 6. Effect of the Invention

[0034] The complex of the present invention can bind strongly and selectively to the higher-order structure of RNA, thereby more effectively inhibiting the transcription and replication of RNA.

[0035] According to the viral RNA transcription inhibitor of the present invention, it is possible to more effectively inhibit the transcription and replication of RNA.

[0036] According to the viral RNA replication inhibitor of the present invention, it is possible to more effectively inhibit RNA replication.

[0037] The pharmaceutical composition of the present invention makes it possible to effectively inhibit the transcription or replication of viral RNA.

[0038] According to the screening method of the present invention, compounds capable of inhibiting RNA virus replication or transcription can be selected with high binding ability and selectivity.

[0039] The kit of the present invention enables the ability of a compound to inhibit the replication or transcription of viral RNA to be evaluated with higher accuracy. [Brief description of the drawings]

[0040] [Figure 1] Schematic diagram showing examples of RNA structures and complexes of the present invention. [Diagram 2] (A) Schematic diagram showing the structure of the promoter region of influenza A virus single-stranded RNA. (B) Various models of the promoter region of influenza A virus single-stranded RNA. [Diagram 3] The sequences of various RNAs used in this example. The regions targeted by the probes are indicated by black dots. [Figure 4](A) Chemical formula of the tFIT-DPQ probe, (B) Structural formula of the peptide nucleic acid of the tFIT-DPQ probe. [Diagram 5] Fluorescence spectra of each probe in the absence and presence of target RNA at pH 5.5 and pH 7.0. (A) tFIT-DPQ probe, (B) control probe. [Figure 6] Fluorescence excitation spectra of each probe in the presence of target RNA at pH 7.0. (A) tFIT-DPQ probe, (B) control probe. [Figure 7] Fluorescence sensing ability of each probe in the absence and presence of each double-stranded RNA at pH 7.0. (A) tFIT-DPQ probe, (B) control probe. [Figure 8] (A) Target RNA sequence, (B) Graph showing fluorescence intensity when tFIT-DPQ probe was bound to the target RNA in Figure 8A. (C) Graph showing RNA concentration-dependent change in fluorescence intensity. (D) No loop RNA sequence, (E) Graph showing fluorescence intensity when tFIT-DPQ probe was bound to the no loop RNA in Figure 8D. (F) Graph showing RNA concentration-dependent change in fluorescence intensity. [Figure 9] Photograph of a gel of polyacrylamide gel electrophoresis to evaluate the ability to inhibit viral RNA replication in vitro. Lane 1: no 53mer (template strand), no compound, lanes 2-11: 10 ng of 53mer added, lane 2: no compound, lanes 3, 6, 9: 5 μM compound added, lanes 4, 7, 10: 15 μM compound added, lanes 5, 8, 11: 50 μM compound added. [Figure 10] Photograph of a gel of polyacrylamide gel electrophoresis to evaluate the ability to inhibit viral RNA transcription in vitro. Lane 1: no vRNP, no compound, lanes 2-11: 20ng vRNP added, lane 2: no compound, lanes 1-11: 1000cpm of cap1-RNA 70mer added, lanes 3, 6, 9: 1.5μM compound added, lanes 4, 7, 10: 3.6μM compound added, lanes 5, 8, 11: 6.0μM compound added. [Figure 11] Evaluation of transcription inhibitory activity in cultured cells using the Dual-luciferase Reporter Assay System. [Figure 12] Scheme of the FID assay using the tFIT-DPQ probe. [Figure 13] Changes in fluorescence intensity depending on the concentration of test compounds. (A) Chemical formula and Kd of DPQ, (B) Graph of fluorescence intensity versus concentration when DPQ is used, (C) Chemical formula and Kd of neomycin, (D) Graph of fluorescence intensity versus concentration when neomycin is used, (E) Chemical formula and Kd of kanamycin, (F) Graph of fluorescence intensity versus concentration when kanamycin is used. RNA 1μM, DPQ-PNA 100nM, test compound concentrations 0.05-6000μM, NaCl 100mM, sodium phosphate 10mM, EDTA 1.0mM, pH7.0, 25℃. Analysis was performed at λex = 515nm, 534nm. Errors are standard deviations obtained from three independent experiments. [Figure 14] (A) Structural formula of probe 2; (B) Chemical formula of probe 2. [Figure 15] Fluorescence spectra of probe 2 in the absence and presence of target RNA at pH 5.5 and pH 7.0. [Figure 16] Fluorescence sensing ability of probe 2 in the absence and presence of each double-stranded RNA at pH 7.0. [Figure 17] (A) Graph showing the fluorescence intensity when probe 2 was bound to the target RNA in Fig. 8 A. (B) Graph showing the RNA concentration-dependent change in fluorescence intensity. [Figure 18] (A) Structural formula of probe 3; (B) Chemical formula of probe 3. [Figure 19] Fluorescence spectra of probe 3 in the absence and presence of target RNA at pH 5.5 and pH 7.0. [Figure 20] Fluorescence sensing ability of probe 3 in the absence and presence of each double-stranded RNA at pH 7.0. [Figure 21](A) Graph showing the fluorescence intensity when probe 3 was bound to the target RNA in Fig. 8A. (B) Graph showing the RNA concentration-dependent change in fluorescence intensity. [Figure 22] Photograph of a gel of polyacrylamide gel electrophoresis to evaluate the ability to inhibit viral RNA transcription in vitro. Lane 1: no vRNP, no compound, lanes 2-11: 20ng vRNP added, lane 2: no compound, lanes 1-11: 1000cpm of cap1-RNA 70mer added, lanes 3, 6, 9: 0.3μM compound added, lanes 4, 7, 10: 1μM compound added, lanes 5, 8, 11: 3μM compound added. [Figure 23] (A) Structural formula of probe 4, (B) Comparison of the thiazole orange (TO) spacer in probe 4 with that in probe 1, (C) Chemical formula of probe 4. [Figure 24] Fluorescence spectra of probe 2 in the absence and presence of target RNA at pH 5.5 and pH 7.0. [Diagram 25] (A) Graph showing the fluorescence intensity when probe 3 was bound to the target RNA in Fig. 8A. (B) Graph showing the RNA concentration-dependent change in fluorescence intensity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] According to a first aspect of the present invention, there is provided a complex targeting an RNA, the RNA extending from the 5' end to the 3' end, comprising a first nucleic acid sequence, a second nucleic acid sequence located on the 3' end side of the first nucleic acid sequence and forming a double strand with the first nucleic acid sequence, and an internal loop formed between the double strand, and the sequence from the 5' end to the 8th or 9th base of the first nucleic acid sequence is a target nucleic acid sequence that is complementarily bound by the complex. The complex comprises a PNA (peptide nucleic acid) strand that binds complementarily to the above-mentioned target nucleic acid sequence of RNA and has the same strand length as the target nucleic acid sequence, 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ), and a linker that connects the PNA strand and the DPQ.

[0042] The first and second nucleic acid sequences of the RNA form a double strand by hydrogen bonds between complementary base pairs, and the PNA strand of the complex binds to the target nucleic acid sequence in the first nucleic acid sequence of the RNA in a sequence-selective manner with the double strand of the RNA. In addition, the DPQ of the complex binds to the internal loop of the RNA. This allows the complex to exhibit excellent binding strength and binding selectivity to the target RNA.

[0043] FIG. 1 shows an example of RNA and the complex of the present invention. RNA 10 extends from the 5' end to the 3' end, and includes a first nucleic acid sequence 11 and a second nucleic acid sequence 12 located on the 3' end side of the first nucleic acid sequence 11 and forming a double strand with the first nucleic acid sequence 11, and an internal loop 13 is formed between the double strand formed by the first nucleic acid sequence 11 and the second nucleic acid sequence 12. Complex 20 includes PNA strand 21, 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ) 22, and a linker 23 connecting the PNA strand 21 and DPQ 22, and a portion of the PNA strand 21 binds complementarily to a target nucleic acid sequence 14 from the 5' end to the 9th base of the first nucleic acid sequence 11 of RNA. In addition, DPQ 22 of complex 20 binds to the internal loop of RNA 10. Therefore, complex 20 exhibits excellent binding strength and binding selectivity to the target RNA 10.

[0044] The RNA is not particularly limited as long as it has a double-stranded structure and an internal loop structure, but is preferably influenza A virus RNA.

[0045] Influenza A virus particles have a membranous structure called an envelope, and form spherical or thread-like structures with a diameter of 80-130 μm or 1-2 μm. Large amounts of hemagglutinin (HA) and neuraminidase (NA) are present on the membrane surface, M2 protein penetrates the membrane, and M1 protein is present inside the membrane. The viral genome is composed of eight different ribonucleoprotein complexes (RNPs), and each RNP contains a single-stranded negative-sense RNA.

[0046] In influenza A virus, all eight genomic RNAs (NA, HA, M1, M2, PB1, PB2, PA, NP) have non-translated regions at the 5' and 3' ends. Except for the C4 mutation, in which the fourth uracil from the 3' end is mutated to cytosine, which is occasionally seen in segments 4, 5, and 8, the 13 bases from the 5' end and the 12 bases from the 3' end are conserved in all eight segments and are called the promoter region (Figure 2A). It is known that the bases at the 5' end and 3' end of the promoter region interact with each other, contributing to the stabilization of polymerase binding and being necessary for RNA synthesis. A coding region exists between the promoter region at the 5' end and the promoter region at the 3' end.

[0047] The higher-dimensional structure of the promoter region is still unclear, and various models have been proposed (Front. Microbiol., 2018,9,559). Among them, the panhandle model is the most supported by the NMR structure of a model sequence in which two conserved sequences with the most base pairs are chemically synthesized and connected by a loop, and the results of biophysical analysis of isolated promoter regions in solution (Figure 2B). This model has an A-type duplex structure containing a CA mismatch formed by the binding of the 5'-end base and the 3'-end base, and an (AA)-U internal loop, with a bend of 46±10°. These structures are thought to be important for RNA polymerase recognition.

[0048] Preferably, the first nucleic acid sequence of the RNA is all or a portion of the promoter region on the 5' end of the influenza A virus RNA, and the second nucleic acid sequence is all or a portion of the promoter region on the 3' end of the influenza A virus RNA.

[0049] The first nucleic acid sequence of the RNA molecule is preferably all or part of the 13 bases from the 5' end of the RNA, and the target nucleic acid sequence is the 8th or 9th base from the 5' end of the first nucleic acid sequence. The second nucleic acid sequence of the RNA molecule is preferably all or part of the 12 bases from the 3' end of the RNA molecule.

[0050] When the RNA targeted by the complex of the first embodiment is influenza A virus RNA, the first nucleic acid sequence of the RNA is preferably a nucleic acid sequence of 12 or 13 bases from the 5' end of the RNA corresponding to the promoter on the 5' end side. The second nucleic acid sequence of the RNA is preferably a nucleic acid sequence of 11 or 12 bases from the 3' end of the RNA corresponding to the promoter on the 3' end side. Since 13 bases from the 5' end and 12 bases from the 3' end of the promoter region of influenza A virus are highly conserved, the complex of the first embodiment, which is equipped with a PNA strand capable of binding to the double stranded RNA formed by these portions, is less susceptible to the effects of mutations in the viral RNA and can maintain a high binding ability to RNA.

[0051] Compared to other artificial nucleic acids, PNA strands are superior in that they have high enzyme resistance against nucleases and proteases, have chemical stability without charge repulsion, and are stable in vivo. For these reasons, they were adopted as a component of the complex of the first embodiment due to their excellent properties.

[0052] A PNA strand is an artificial molecule in which N-(2-aminoethyl)glycine is bonded by an amide bond in place of sugar in a nucleic acid (DNA or RNA). The PNA strand of the complex is not particularly limited as long as it is a PNA capable of binding to a double strand of RNA, but preferably has a sequence that is completely complementary or complementary except for one base to the bases of the target nucleic acid sequence of the first nucleic acid sequence of the RNA strand. The base length of the PNA strand is preferably 8, 9, or 10 (when a label molecule is inserted between the bases of the PNA, the number of the label molecule is counted as one base).

[0053] In a preferred embodiment, the PNA strand of the complex has 8, 9 or 10 bases that are fully complementary or have all but one base complementary to the bases of the target nucleic acid sequence of the first nucleic acid sequence of the RNA, such that the PNA strand of the complex can more selectively bind to the RNA.

[0054] In another preferred embodiment, the PNA strand of the complex has a sequence that is completely complementary to 8 or 9 bases from the 5' end of the RNA, or a sequence that is complementary except for one base, and the base length of the PNA is 8, 9, or 10. By configuring the PNA strand of the complex in this way, the complex can more selectively bind to the RNA.

[0055] Labeling molecules that can be bound to PNA strands include fluorescent dyes, enzymes, radioactive labels, etc. Labeling molecules can be attached to the 5' end of the PNA strand, or can be incorporated as intercalators by substituting bases in the PNA. As used herein, "intercalators" refer to compounds that are inserted between the nucleic acid bases of the PNA strand.

[0056] Examples of the labeling molecule attached to the 5' end of the PNA strand include, for example, fluorescein and its derivatives (e.g., fluorescein-5(6)-isothiocyanate (FITC), fluorescein-4-isothiocyanate, tetrachlorofluorescein, hexachlorofluorescein, tetrabromosulfonefluorescein (TBSF) and their derivatives), EDANS (5-(2-aminoethyl)amino-1-naphthalenesulfonic acid), 6-JOE, Alexa Fluor 488 (Molecular Probes), Alexa Fluor 532 (Molecular Probes), Cy3 (Amersham Biosciences), Cy5 (Amersham Biosciences), Pacific Examples of such an antibody include, but are not limited to, BODIPY Blue (Molecular Probes), Texas Red (Molecular Probes), BODIPY-FL (Molecular Probes), BODIPY-FL / C3 (Molecular Probes), BODIPY-FL / C6 (Molecular Probes), BODIPY-5-FAM (Molecular Probes), BODIPY-TMR (Molecular Probes), BODIPY-TR (Molecular Probes), BODIPY-R6G (Molecular Probes), BODIPY564 (Molecular Probes), and BODIPY581 (Molecular Probes).

[0057] Examples of labeling molecules incorporated into the PNA chain include thiazole orange, acridine dyes (e.g., acridine orange), ethidium compounds (e.g., ethidium bromide, ethidium homodimer 1, ethidium homodimer 2, ethidium bromide monoazide (EMA), dihydroethidium), iodine compounds (e.g., propidium iodide, hexidium iodide), 7-aminoactinomycin D (7-AAD), cyanine monomer dyes (thiazole orange, quinoline blue, e.g., PO-PRO-1, BO-PRO-1, YO-PRO-1, TO-PRO-1, JO-PRO-1, PO-PRO-3, LO-PRO-1, BO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5, all from Molecular Probes), cyanine dimer dyes (e.g., POPO-1, BOBO-1, YOYO-1, TOTO-1, Examples of dyes include, but are not limited to, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3 (all manufactured by Molecular Probes), SYTOX dyes (e.g., SYBR Gold, SYBR Green I and SYBR Green II, SYTOX Green, SYTOX Blue, SYTOX Orange (all manufactured by Molecular Probes), and GelRed (Wako Pure Chemical Industries, Ltd.).

[0058] A preferred labeling molecule is a fluorescent dye, and as the fluorescent dye, any known fluorescent dye for labeling nucleic acids can be used.

[0059] Preferred fluorescent dyes are cyanine dyes, or fluorescein and its derivatives. The cyanine dye is preferably methine cyanine. At least one PNA constituting the PNA chain is substituted with methine cyanine. Methine cyanine exhibits significant fluorescence amplification when incorporated into PNA as a pseudobase. Examples of methine cyanine include monomethine cyanine and trimethine cyanine. Examples of methine cyanine include thiazole orange (TO), quinoline blue (QB), and thiazole red (TR).

[0060] The binding site of the labeling molecule to the PNA strand is not particularly limited, but it is preferable that in the double strand formed by the first and second nucleic acid sequences of RNA, the PNA on the PNA strand at the position complementary to the RNA on the first nucleic acid sequence where base pair mismatch occurs is replaced with the labeling molecule. Such a replacement position may correspond to the third or fourth position from the 5' end of the PNA strand. In this case, the binding of the complex to the RNA can be detected with a stronger signal. Preferably, the base sequence of the PNA strand is N-terminus-NTCNTCTTT-C-terminus (1) In formula (1), the first N is no base or C, and the fourth N is G, C, A, T, or methinecyanine.

[0061] Such a PNA strand configuration allows specific recognition of the target nucleic acid sequence of the first nucleic acid sequence of RNA, and allows detection of the binding of the complex to RNA with a stronger signal. A preferred embodiment of the PNA strand incorporating a labeling molecule is (i) a PNA strand having a sequence that is completely complementary to 8 or 9 bases from the 5' end of the RNA, or a sequence that is complementary except for one base, the PNA has a base length of 8 bases, the labeling molecule is at the third position from the N-terminus of the PNA strand, and the labeling molecule is a cyanine dye, or fluorescein and its derivatives, or (ii) a PNA strand having a sequence that is completely complementary to 8 or 9 bases from the 5' end of the RNA, or a sequence that is complementary except for one base, the PNA has a base length of 9 bases, the labeling molecule is at the fourth position from the N-terminus of the PNA strand, and the labeling molecule is a cyanine dye, or fluorescein and its derivatives. In (i) and (ii), the RNA is preferably the RNA of influenza A virus.

[0062] A particularly preferred PNA strand is a PNA called tFIT probe (triplex-forming forced intercalation of thiazole orange) developed by the present inventors (J. Am. Chem. Soc., 2016, 138, 9397-9400). This probe is designed by introducing an intercalator, thiazole orange (TO), into the interior of the triplex-forming PNA, so that TO is forcibly intercalated as the triplex is formed. The tFIT probe shows almost no fluorescence response by itself, but shows a clear fluorescence response in the presence of target double-stranded RNA (Light-up factor; I / I0>100), and is the first probe that can directly sense the RNA double-stranded structure, which is one of the higher-order structures, in a sequence-selective manner. The tFIT probe is represented by the following formula (I).

[0063] [ka]

[0064] DPQ (6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) was discovered in 2014 as a small molecule that binds to the internal loop of influenza A by the same research group that revealed that the promoter region of influenza A RNA in solution forms a panhandle-like A-type duplex containing an AC mismatch and an (AA)-U internal loop (Chem. Commun., 2014, 50, 368-370). DPQ is represented by the following formula (II):

[0065] [ka]

[0066] The oxygen of the two methoxy groups in the DPQ molecule forms hydrogen bonds with the adenine in the internal loop of the promoter region of the RNA and the cytosine above it, and DPQ alone binds to the promoter region with a dissociation constant of 50.5 μM. DPQ has antiviral activity, but its activity is inferior to that of existing antiviral drugs.

[0067] DPQ may have a substituent on the piperazinyl group for linking with a linker. For example, a carboxylic acid may be added to the secondary amine of piperazine. DPQ and a carboxylic acid may be reacted to add a carboxyl group as a substituent to DPQ, and then the amino group of the linker and the carboxyl group added to DNP may be bonded.

[0068] The type of linker is not particularly limited as long as it connects PNA and DPQ, and a person skilled in the art can select an appropriate linker. For example, organic molecules having an amino group are well known as linkers. An organic molecule refers to a molecule having a carbon atom. Examples of organic molecules having an amino group include alkylamines (particularly those having a primary amino group bonded to a linear carbon chain, which may have an ether bond in the hydrocarbon chain), carbamates, methylene, amino acids, peptides, sulfonamides, siloxanes, silanes, carbonates, carboxymethyl, thioethers, ethylene oxides, sulfonates, oximes, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, methylenecarbonylamino, succinimidyl, polyalkylene glycols, maleimides, polysaccharides, disulfides, triazoles, and the like. Examples of amino acids constituting the amino acid linker or peptide linker include lysine, arginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, and ornithine.

[0069] The linker is usually linked to the PNA strand at the 3' end of the PNA strand. The linking mode between the PNA strand and the linker can be an amide bond (including a peptide bond), an ester bond, etc., and is preferably, but not limited to, an amide bond.

[0070] In addition, the linker is linked to DPQ via a functional group other than the linking group to the PNA chain. The linker and DPQ can be linked by an amide bond (including a peptide bond), an ester bond, etc., and the amide bond is preferred, but is not limited thereto. An example of the linkage between the linker and DPQ is to react DPQ with a carboxylic acid to add a carboxyl group to DPQ as a substituent, and then bond the amino group of the linker to the carboxyl group added to DPQ.

[0071] The inventors considered that the 5'-end of the promoter region of the RNA is a sequence rich in purine bases, and that it forms a double strand with the 3'-end, and therefore it is a sequence that can be targeted by a tFIT probe consisting of a PNA strand. Furthermore, since there is an internal loop, which is a characteristic higher-order structure, near the double strand, and DPQ binding to this has been reported recently, it was considered possible to develop a complex probe with high selectivity and binding strength.

[0072] Surprisingly, the present inventors have found that the complex according to the first aspect of the present invention inhibits RNA transcription and replication to a greater extent than DPQ alone.

[0073] The binding ability of the complex according to the first aspect of the present invention to RNA was measured using the dissociation constant K d It can be evaluated by:

[0074] Dissociation constant K of the complex of the first embodiment for RNA d is preferably 100 nM or less, more preferably 50 nM or less.

[0075] The complex of the first aspect of the present invention inhibits the transcription and replication of viral RNA and is therefore useful as a viral RNA transcription inhibitor and a viral RNA replication inhibitor, and is also useful as an active ingredient of a pharmaceutical composition for inhibiting the transcription or replication of viral RNA and a pharmaceutical composition for preventing or treating viral infection.

[0076] According to a second aspect of the present invention, there is provided a viral RNA transcription inhibitor comprising the complex according to the first aspect of the present invention. The subject to which the viral RNA transcription inhibitor according to the second aspect of the present invention is administered is a mammal, for example, a human or a non-human mammal. In the following context, non-human mammals include monkeys, pigs, cows, horses, sheep, dogs, cats, rabbits, rats, mice, etc.

[0077] The route of administration of the viral RNA transcription inhibitor of the second aspect of the present invention is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include intravenous administration, intradermal administration, subcutaneous administration, inhalation administration, transdermal administration, transmucosal administration, and rectal administration. Furthermore, a known encapsulating agent that enhances the persistence and membrane permeability of the viral RNA transcription inhibitor may be used in combination. Examples of such encapsulating agents include liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, and derivatives thereof.

[0078] The dosage of the viral RNA transcription inhibitor can be appropriately adjusted according to the condition of the subject, and a preferred amount can be used. For example, the dosage can be in the range of 0.001 to 100 mg / kg, preferably 0.1 to 10 mg / kg, but is not limited thereto.

[0079] According to a third aspect of the present invention, there is provided an inhibitor of viral RNA replication, comprising the complex according to the first aspect of the present invention. The subject to which the inhibitor of viral RNA replication according to the third aspect of the present invention is administered is a mammal, for example a human or a non-human mammal.

[0080] The route of administration of the viral RNA replication inhibitor of the third aspect of the present invention is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include intravenous administration, intradermal administration, subcutaneous administration, inhalation administration, transdermal administration, transmucosal administration, and rectal administration. Furthermore, a known encapsulating agent that enhances the persistence and membrane permeability of the viral RNA replication inhibitor may be used in combination. Examples of such encapsulating agents include liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, and derivatives thereof.

[0081] The dosage of the viral RNA replication inhibitor can be appropriately adjusted according to the condition of the subject, and a preferred amount can be used. For example, the dosage can be in the range of 0.001 to 100 mg / kg, preferably 0.1 to 10 mg / kg, but is not limited thereto.

[0082] According to a fourth aspect of the present invention, there is provided a pharmaceutical composition for inhibiting transcription or replication of viral RNA, comprising the complex according to the first aspect of the present invention. In the fourth aspect, the complex is preferably free of a labeling molecule. The subject to which the pharmaceutical composition according to the fourth aspect of the present invention is administered is a mammal, for example, a human or a non-human mammal.

[0083] The administration route of the pharmaceutical composition for inhibiting the transcription or replication of viral RNA according to the fourth aspect of the present invention is not particularly limited, and may be orally or parenterally. Examples of parenteral administration include intravenous administration, intradermal administration, subcutaneous administration, inhalation administration, transdermal administration, transmucosal administration, and rectal administration.

[0084] The dosage of the complex in the pharmaceutical composition can be appropriately adjusted according to the condition of the subject, and a preferred amount can be used. For example, the complex can be administered in the range of 0.001 to 100 mg / kg, preferably 0.1 to 10 mg / kg, but is not limited thereto.

[0085] The pharmaceutical composition may contain a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier is an inactive substance that facilitates administration of the RNA of the complex to a subject. As the pharma- ceutical acceptable carrier, in addition to the known encapsulating agent that enhances the persistence and membrane permeability of the above-mentioned viral RNA replication inhibitor, various conventional organic or inorganic carrier substances are used, and are compounded as excipients, lubricants, binders, disintegrants in solid preparations, solvents, solubilizing agents, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid preparations. In addition, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as necessary.

[0086] According to a fifth aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a viral infection, comprising the complex according to the first aspect of the present invention. In the fifth aspect, the complex is preferably free of a labeling molecule. The subject to which the pharmaceutical composition according to the fifth aspect of the present invention is administered is a mammal, for example a human or a non-human mammal.

[0087] The administration route of the pharmaceutical composition for inhibiting the transcription or replication of viral RNA according to the fifth aspect of the present invention is not particularly limited, and may be orally or parenterally. Examples of parenteral administration include intravenous administration, intradermal administration, subcutaneous administration, inhalation administration, transdermal administration, transmucosal administration, and rectal administration.

[0088] The therapeutically effective amount or dosage of the complex in the pharmaceutical composition can be appropriately adjusted according to the condition of the subject, and a preferred amount can be used. For example, the complex can be administered in the range of 0.001 to 100 mg / kg, preferably 0.1 to 10 mg / kg, but is not limited thereto. The "therapeutically effective amount" is the amount of the complex that can provide the desired therapeutic result (e.g., reduction or elimination of viral infection).

[0089] The pharmaceutical composition may contain a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier is an inactive substance that facilitates administration of the RNA of the complex to a subject. As the pharma- ceutical acceptable carrier, in addition to the known encapsulating agent that enhances the persistence and membrane permeability of the viral RNA replication inhibitor described above, various conventional organic or inorganic carrier substances are used, and are compounded as excipients, lubricants, binders, disintegrants in solid preparations, solvents, solubilizing agents, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid preparations. In addition, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as necessary.

[0090] According to a sixth aspect of the present invention there is provided a method of inhibiting transcription or replication of RNA in vitro or in a non-human mammalian subject, the method comprising contacting the RNA with a complex of the first aspect of the present invention.

[0091] The RNA extends from the 5' end to the 3' end and comprises a first nucleic acid sequence, a second nucleic acid sequence located 3' closer to the first nucleic acid sequence and forming a double strand with the first nucleic acid sequence, and an internal loop formed between the double strands.

[0092] The complex comprises a PNA strand that binds complementarily to a target nucleic acid sequence from the 5' end of the first nucleic acid sequence of the RNA to the 8th or 9th base and has the same chain length as the target nucleic acid sequence, 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ), and a linker that connects the PNA strand and DPQ.

[0093] In the in vitro method, a sample containing RNA or a sample suspected of containing RNA is contacted with the complex of the first aspect of the present invention. The sample may be, for example, a cultured cell or a biological sample (blood, saliva, mucosa, urine, etc.) taken from a subject infected with or suspected of being infected with a virus. Inhibition of RNA transcription or replication can be detected by a method for measuring transcription or inhibition known in the art.

[0094] According to a seventh aspect of the present invention, there is provided a screening method for a compound capable of inhibiting RNA replication or transcription, comprising the steps of: binding RNA to the complex of the first aspect of the present invention to form a conjugate; contacting the conjugate with a test compound; and measuring each signal value obtained from the conjugate before and after the contact.

[0095] When each signal value obtained from the conjugate after contacting the conjugate with the test compound is lower than each signal value obtained from the conjugate before contacting the conjugate with the test compound, the test compound can be determined to be a hit compound.

[0096] In the seventh embodiment, the complex of the first embodiment is labeled with a fluorescent dye, and when the fluorescence intensity obtained from the complex of the first embodiment after contacting the complex of the RNA with a test compound is lower than the fluorescence intensity obtained from the complex before contacting the complex with the test compound, it can be determined that the binding of the test compound to the RNA is stronger than the binding of the RNA to the complex. In this case, the test compound can be determined to be a replication or transcription inhibitor for RNA.

[0097] According to an eighth aspect of the present invention there is provided a kit for assessing the ability of a compound to inhibit viral RNA replication or transcription, the kit comprising a complex according to the first aspect of the present invention.

[0098] In one embodiment, the kit of the ninth aspect of the invention may comprise instructions (e.g. instructions on how to process samples, perform the test, interpret the results, how to use the conjugates of the first aspect of the invention and / or how to store the conjugates of the first aspect of the invention), one or more reference samples, a buffer for dissolving the conjugates of the first aspect of the invention and / or other reagents required to perform the test.

[0099] The kit may further comprise one or more containers for separately housing some of the individual components or reagents.

[0100] According to a ninth aspect of the invention there is provided a kit for inhibiting viral RNA transcription or replication and / or for treating a viral infection in a mammal comprising a complex according to the first aspect of the invention.

[0101] In one embodiment, the kit according to the ninth aspect of the invention may comprise instructions (e.g. instructions on how to carry out the test, interpret the results, how to use the conjugate of the first aspect of the invention and / or how to store the conjugate of the first aspect of the invention), a buffer for dissolving the conjugate of the first aspect of the invention, and / or other reagents required to carry out the test.

[0102] The kit may further comprise a syringe, needle, applicator, etc. for administering the complex of the first aspect of the present invention to a subject.

[0103] According to a tenth aspect of the present invention there is provided a kit for assessing viral infection comprising a complex according to the first aspect of the present invention.

[0104] According to an eleventh aspect of the present invention, there is provided an in vitro test agent comprising the conjugate according to the first aspect of the present invention.

[0105] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. EXAMPLES

[0106] In this example, a fluorescent peptide nucleic acid probe targeting the promoter region of influenza A virus RNA was designed and synthesized, and its function was evaluated. Example 1 1. Preparation of RNA Figure 3 shows the sequences of various RNAs used in this example. The RNAs were synthesized by methods well known to those skilled in the art. The target sequence (target RNA) is a model sequence of a hairpin-loop structure including the influenza A virus RNA promoter region (12 bases at the 3' end and 13 bases at the 5' end). The part of the target RNA surrounded by a dotted line in Figure 3 is the actual sequence, and this model sequence is often used to investigate the characteristics of the promoter region. This target RNA is known to form a double strand with a panhandle-like A-type structure and to have an internal loop structure (Chem.Commun., 2014, 50, 368-370.). In addition, a sequence in which the fourth uracil from the 3' end of the target RNA is changed to cytosine, which is the only naturally occurring mutant, was also used to evaluate the probe (natural mutant RNA in Figure 3). To evaluate the binding properties of the probe, we also prepared a sequence in which the internal loop structure in the sequence was artificially eliminated (no loop RNA), and a sequence in which the AC mismatch and the GU wobble base pair in the sequence were artificially altered to be completely complementary (model RNA). Single-stranded RNA (3'pyrimidine and 5'purine) was also prepared by extracting only the 3' and 5' ends of the promoter region.

[0107] The nucleic acid sequences of Target RNA, Natural Mutant RNA, No Loop RNA, Model RNA, 3'Pyrimidine, and 5'Purine are shown in SEQ ID NOs: 1 to 6, respectively. Target RNA: 5'-GAGUAGAAACAAGGCUUCGGCCUGCUUUUGCU-3' (SEQ ID NO: 1) Natural mutant RNA: 5'-GAGUAGAAACAAGGCUUCGGCCUGCUUUCGCU-3' (SEQ ID NO: 2) No loop RNA: 5'-GAGUAGAAACAAGGCUUCGGCCUUGCUUUUGCU-3' (SEQ ID NO: 3) Model RNA: 5'-GAGUAGAAACAAGGCUUCGGCCUGUUUCUACU-3' (SEQ ID NO: 4) 5'Purine: 5'-GAGUAGAAACAAGG-3' (SEQ ID NO:5) 3'Pyrimidine: 5'-CCUGCUUUUGCU-3' (SEQ ID NO: 6)

[0108] 2. Design of the probe (probe 1) Focusing on the fact that the promoter region of influenza A virus RNA forms a double strand with an internal loop, we attempted to design a PNA-DPQ complex probe that has both high binding strength and selectivity. The inventors considered that since the 5'-terminal region of the promoter region has a purine-rich sequence, it would be possible to recognize it with a tFIT probe (J. Am. Chem. Soc., 2016, 138, 9397-9400) that utilizes Hoogsteen-type hydrogen bonds. Furthermore, since a small molecule (DPQ) that binds to the internal loop of the promoter region has been discovered (Chem. Commun., 2014, 50, 368-370), the inventors considered that by incorporating this into the tFIT probe, it would be possible to develop a probe that can recognize the higher-order structure of the promoter region as it is.

[0109] Of the PNA-DPQ probes, PNA is mainly synthesized by solid-phase synthesis. For binding with PNA, synthesis of a carboxylic acid derivative of DPQ is essential. In DPQ, the methoxy groups at positions 6 and 7 form hydrogen bonds with the nucleic acid bases in the internal loop of the RNA molecule, so it is necessary to extend the carboxylic acid from other positions. Therefore, a carboxylic acid was added to the secondary amine of piperazine at position 2. DPQ-COOH was newly synthesized according to the following scheme 1.

[0110] Specifically, potassium carbonate (1.43g, 10.5mmol) was added to an acetonitrile solution containing DPQ (1.65g, 5.7mmol) and ethyl-2-(p-toluenesulfonyloxy)aqcetate (0.99g, 4.0mmol) synthesized according to J.Med. Chem., 2016, 59 2151-2162. The solution was heated to reflux (90°C) for 30 hours with a stirrer, and then immediately filtered. The filtrate was concentrated under reduced pressure to obtain the crude DPQ-COOEt product.

[0111] The obtained crude product was dissolved in water / THF (60 mL, 3:1 v / v) and cooled on ice. 2.9 M aqueous sodium hydroxide solution was added and stirred for 5 minutes. After stirring at room temperature for 30 minutes, 6 M hydrochloric acid solution was added while cooling on ice and reacted for 1 hour. The solution was concentrated under reduced pressure to obtain the DPQ-COOH crude product. This was used for the linking reaction with PNA.

[0112] [ka]

[0113] In PNA, the N-terminus corresponds to the 5'-terminus of RNA, and the C-terminus corresponds to the 3'-terminus. In the case of duplexes formed between PNA and single-stranded RNA through Watson-Crick hydrogen bonds, antiparallel orientation is preferred, but duplexes can also be formed in parallel orientation. On the other hand, triplexes formed through Hoogsteen hydrogen bonds with double-stranded RNA, with PNA as the third strand, have only been reported in parallel orientation with PNAs containing only thymine and cytosine, so when designing a PNA probe for a target RNA duplex, it must be designed to be parallel to the target purine sequence.

[0114] tFIT as a PNA was synthesized as described in J. Am. Chem. Soc., 2016, 138, 9397-9400.

[0115] In connecting tFIT and DPQ-COOH, the inventors condensed DPQ-COOH to the primary amine of the lysine side chain, since lysine is usually bound to the tFIT probe to improve water solubility and binding strength by electrostatic interaction with the phosphate group. The structural formula (formula (III)) of the tFIT-DPQ probe in which tFIT is bound to DPQ-COOH via lysine, and the structural formula (formula (IV)) of the control probe in which the primary amine of the lysine side chain is acetylated without a DPQ unit are shown below. Thiazole orange (TO), a fluorescent dye in the tFIT probe, was arranged so as to face uracil in the consecutive purine bases of the target RNA. The chemical formula and structural formula of the obtained tFIT-DPQ probe (probe 1) are shown in Figures 4(A) and (B), respectively.

[0116] [ka]

[0117] 3. Fluorescence Measurement The interaction between the probe and RNA was analyzed by fluorescence response.

[0118] The RNA and probe concentrations were both set to 1.0 μM in a buffer solution (pH 5.5, 25° C.) containing 100 mM NaCl, 10 mM sodium acetate, and 1.0 mM EDTA, and fluorescence was measured.

[0119] The conditions of the fluorescence spectrometer were λex = 515 nm, Ex band: 3 nm, Em band: 3 nm, sensitivity: Medium, scan rate 100 nm / min, response: 0.5 sec, 3 × 3 mm quartz cell (JASCO Corporation FP-6500).

[0120] As shown in Figure 5A and B, when RNA was not present, no fluorescence was emitted, but when target RNA was present, a light-up type fluorescence was observed, which shows a fluorescent response. This is a response similar to that of conventional tFIT probes, and it is presumed that the fluorescence is emitted according to the double-stranded sequence. Interestingly, this light-up response was also observed under neutral conditions of pH 7.0 (buffer containing 100 mM NaCl, 10 mM sodium phosphate, 1.0 mM EDTA, 25°C).

[0121] To further investigate this phenomenon, we carefully observed the shapes of the fluorescence and excitation spectra of each probe upon binding to the target RNA at pH 7.0 (Fig. 6A, B).

[0122] Fluorescence was measured in a buffer solution (pH 7.0, 25°C) containing 100 mM NaCl, 10 mM sodium phosphate, and 1.0 mM EDTA, with both RNA and probe concentrations at 100 nM. The fluorescence spectrometer conditions were λex = 515 nm, λem = 540 nm, Ex band: 5 nm, Em band: 5 nm, sensitivity: High, Scan rate 100 nm / min, response: 0.5 sec, and 3 × 3 mm quartz cell (FP-6500).

[0123] As shown in Figure 6A, the symmetry of the fluorescence and excitation spectrum of TO, the fluorescence response site, was maintained in the tFIT-DPQ probe, suggesting that the probe and RNA were bound in a 1:1 ratio. On the other hand, the symmetry of the fluorescence and excitation spectrum was lost in the control probe without DPQ (Figure 6B), suggesting that two molecules of the probe were bound to one RNA molecule.

[0124] Although not wishing to be bound by theory, a job plot in which the total molar concentration of the probe and RNA was kept constant and their molar fractions were changed suggested that the binding mode between the tFIT-DPQ probe and the target RNA was triplex formation based on Hoogsteen base pairing or invasion based on Watson-Crick base pairing, whereas the binding mode between the control probe and the target RNA was triplex invasion, in which another PNA molecule forms a triplex through Hoogsteen-type hydrogen bonds in the new double strand formed by invasion (data not shown).

[0125] 4. Effects of RNA sequences To evaluate the structural selectivity of each probe at pH 7.0, we observed the fluorescence response of the various RNAs shown in Figure 3 with the tFIT-DPQ probe (Figure 7A) or control probe (Figure 7B). The area of ​​the fluorescence spectrum (520-700 nm) normalized to the response to the target RNA as 1 is summarized in a bar graph. The tFIT-DPQ probe gave a selective fluorescence response to the target RNA (Figure 7A). In particular, the response to no loop RNA, which does not have an internal loop structure, was reduced, suggesting that the DPQ probe recognizes the internal loop. The response was also reduced with the natural mutant, which is presumed to be due to the binding mode of the tFIT-DPQ probe and the natural mutant RNA being triple invasion (data not shown).

[0126] On the other hand, no clear difference was observed between the target RNA and the natural mutant RNA or no loop RNA when using the control probe (Fig. 7B). In addition, the response to complementary single strands was increased, whereas it was almost nonexistent when using the tFIT-DPQ probe. This suggests that by linking DPQ to PNA, we were able to obtain internal loop selectivity and suppress the response to single strands.

[0127] The response to the model RNA was reduced for all probes, which was thought to be related to the fact that the model RNA formed a tighter duplex (completely complementary duplex) than the target RNA.

[0128] 5. Calculation of dissociation constants by fluorescence titration experiments The binding strength of the tFIT-DPQ probe to the target RNA was calculated and its recognition ability of the internal loop structure was quantitatively evaluated by a fluorescence titration experiment (Figure 8).

[0129] A buffer solution (pH 7.0, 25° C.) containing 100 mM NaCl, 10 mM sodium phosphate, and 1.0 mM EDTA was used, the RNA concentration was varied from 0 to 800 nM, and the probe concentration was set to 100 nM, and fluorescence was measured.

[0130] The fluorometer conditions were λex = 515 nm, Ex band: 3 nm, Em band: 3 nm, sensitivity: High, response: 1.0 sec, 3 × 3 mm quartz cell (FP-6500), and analysis at 534 nm.

[0131] First, the concentration dependence of the obtained fluorescence intensity was analyzed using a 1:1 binding fitting equation, and it was found that the tFIT-DPQ probe had a strong binding affinity to the target RNA with a dissociation constant of 29 ± 3.1 nM (n = 3) (Figure 8A-C). This suggested that the dissociation constant (K d = 50.5 μM, see Chem. Commun., 2014, 50, 368-370), which is an improvement of more than three orders of magnitude. Furthermore, since the tFIT-DPQ probe binds to no loop RNAs that do not have internal loops in a similar manner to the target RNA, we performed a titration experiment with no loop RNA and found that K d The activity was 200 ± 12 nM (n = 3) (Fig. 8D-F), indicating that the DPQ probe recognized the internal loop.

[0132] 6. Evaluation of RNA replication inhibition using viral polymerase Using the tFIT-DPQ probe, the ability of the PNA-DPQ probe to inhibit the replication of influenza A virus RNA in vitro was evaluated. Specifically, vRNP (a complex of viral RNA and polymerase, NP), a 53-mer RNA template (SEQ ID NO: 7) as a catalyst for RNA synthesis, and a probe were added to the solution so that the final solution volume was 25 μL, and a solution with the composition shown in Table 1 was prepared. After incubation at 30°C for 60 minutes, RNA was extracted with phenol / chloroform / ethanol. The extracted RNA was subjected to electrophoresis on a 10% polyacrylamide gel containing 8 M urea, and the amount of RNA produced was evaluated by autoradiography (Figure 9).

[0133] The amount of full-length genomic RNA decreased in a concentration-dependent manner with the PNA-DPQ probe, indicating that it inhibited the RNA replication process of the viral polymerase. On the other hand, even when the concentration of DPQ alone or the control probe (Ctl-PNA) was increased, the amount of RNA generated did not change from that when no compound was added, indicating that replication was not inhibited. This indicates that only the PNA-DPQ probe has RNA replication inhibitory activity. This is thought to be due to the strong binding strength and structural selectivity of the PNA-DPQ probe.

[0134] [Table 1]

[0135] 7. Evaluation of endonuclease activity inhibition using viral polymerase Along with replication, the transcription of viral mRNA (vmRNA) is another important process in the viral multiplication process. Therefore, following the inhibition of the RNA replication process, in this experiment, we evaluated the in vitro transcription inhibition ability of the probe. In the viral transcription process, a process called cap-snatching (Biochemistry, 1997, 36, 5072-5077), which recognizes and cleaves the cap structure of mRNA derived from the host cell, is essential. Cleaving nucleic acids means that the viral polymerase has endonuclease activity, and we specifically evaluated the inhibition ability of this process here.

[0136] Specifically, RNA with a cap structure (cap-1-RNA-70mer, the 70 nucleotide sequence is shown in SEQ ID NO:8), purified viral polymerase, vRNP, and probe were added to the solution so that the final solution volume was 20 μL, and a solution with the composition shown in Table 2 was prepared. The solution was then incubated at 30°C for 60 minutes, and RNA was extracted with phenol / chloroform / ethanol. The extracted RNA was subjected to electrophoresis on a 15% polyacrylamide gel containing 8 M urea, and the amount of cleaved short RNA produced was evaluated by autoradiography (Figure 10).

[0137] A tFIT-DPQ probe was used as the PNA-DPQ probe. It was found that the amount of short RNA generated decreased in a concentration-dependent manner with the PNA-DPQ probe. This indicates that the PNA-DPQ probe inhibits the endonuclease activity of the viral polymerase, which may result in transcription inhibition. On the other hand, the amount of short RNA generated by the control probe and DPQ alone did not change compared to when no compound was added, suggesting that they had no inhibitory activity. Interestingly, this inhibition of the transcription process occurred at a concentration one order of magnitude lower than the inhibition of the replication process mentioned above, indicating that the main process of viral growth inhibition by the PNA-DPQ probe is transcription inhibition.

[0138] [Table 2]

[0139] 8. Evaluation of antiviral activity by reporter assay Since the probe was shown to have the ability to inhibit viral proliferation in vitro, we evaluated whether the probe functions in human cervical cancer cells (HeLa cells) using the Dual-luciferase Reporter Assay System. Here, a DPQ probe fluorescently labeled with FITC (fluorescein 5(6) isocyanate) was newly synthesized to confirm intracellular uptake. The structural formula of the FITC-labeled PNA-DPQ probe is shown in formula (V). The 4th position of PNA was changed to thymine.

[0140] [ka]

[0141] First, cultured HeLa cells attached to a 10 cm diameter dish were detached using trypsin / EDTA, and the cells were collected by centrifugation. The number of cells was counted, and PBS buffer was added to adjust the cell suspension to 3 × 107 cells / mL. To efficiently incorporate the probe into the cells, the probe was introduced into the cells using the Neon (registered trademark) Transfection System. This device is used for electroporation. A tip-type electrode (~10μL) was attached to a Neon pipette, and a mixed solution of the probe and cell suspension was aspirated. Electric pulses were applied to HeLa cells in an electrolyte-containing buffer (E buffer) using the company's recommended protocol (1005 V, 35 ms, 2 pulses), and the cells were transferred to a 12-well culture plate containing 1 mL of MEM (+). The culture plate was incubated at 37°C for 12 hours, and then a fluorescent microscope was used to confirm that the cells had attached to the dish and that the probe had been taken up. The medium was then replaced, and the plasmid was introduced into the cells using a cationic lipid transfection reagent (Gene Juice®). The plasmids introduced were pCAGGS (empty vector) and pHHvNSLuc (expressing Firefly luciferase), 20ng. Since the mRNA expressing Firefly luciferase from Photinus pyralis has the same sequence as the promoter region (it has the promoter region of AGUAGAAACAAGG at the 5' end and UCG(U / C)UUUCGUCC at the 3' end, with the coding region between them), the expression level decreases when the probe inhibits replication and transcription. Renilla luciferase from Renilla reniformis, which is constitutively expressed, was used as an internal control, and the two plasmids were co-transfected to correct the transfection efficiency. After transfection with the plasmids, the cells were incubated at 37°C for 18 hours. The cells were then detached in the same manner as in the first step, collected by centrifugation, and Renilla buffer was added. The cell suspension was subjected to repeated freezing and thawing to prepare a cell lysate. The lysate and its substrate (D-luciferin or coelenterazine) (see Acc. Chem. Res., 1999, 32, 624-630) were added, and chemiluminescence was measured using a photometer. As a result, it was found that the expression of Firefly luciferase was inhibited by about 50% when the probe was added compared to the control when only buffer was added (Figure 11).This suggested that the probe may be capable of inhibiting viral proliferation in vivo.

[0142] 9. Method for screening compounds capable of inhibiting viral transcription or replication A fluorescence indicator displacement assay (also called FID assay) was performed using the tFIT-DPQ probe as an indicator and a small molecule that binds to an existing RNA promoter region as a test compound (Figure 12). 100 nM of the tFIT-DPQ probe was added to 1 μM of the target promoter RNA and incubated at room temperature for 15 minutes, and then the test compound was added and incubated for 15 minutes, after which the fluorescence was measured. As shown in Figures 13(B), (D), and (F), it was found that the fluorescence response derived from thiazole orange (TO) in the probe decreased depending on the concentration of the test compound. The half maximal inhibition concentration (IC50) was calculated from the following formula (VI), and an IC50 was obtained that matched the dissociation constant of each test compound. From these results, it can be said that the tFIT-DPQ probe is useful as an indicator for screening.

[0143]

number

[0144] In the formula, i is the fluorescence intensity, imin is the fluorescence intensity when an excess amount of the test compound is added, imax is the fluorescence intensity in the absence of the test compound, and p is the Hill coefficient.

[0145] Example 2 1. Probe 2 Design Probe 2 was synthesized, which is different from the tFIT-DPQ probe (probe 1) synthesized in Example 1. The structural formula and chemical formula of probe 2 are shown in Figures 14A and 14B, respectively. The structure of probe 2 differs from that of probe 1 in that the first base C next to the NH2 terminus of the PNA strand has been deleted. Probe 2 was synthesized according to "2. Probe design" in Example 1, except that the PNA sequence was changed from that of probe 1.

[0146] 2. Fluorescence Measurement The interaction between probe 2 and RNA was analyzed according to [3. Fluorescence measurement] of Example 1. The reagents used other than probe 2 and their concentrations, and the conditions of the fluorometer were as described in [3. Fluorescence measurement] of Example 1. As shown in Figure 15, when probe 2 was used, no fluorescence was emitted in the absence of RNA, but light-up type fluorescence was observed, which indicates a fluorescent response in the presence of target RNA. This light-up response was observed even under neutral conditions of pH 7.0 (buffer containing 100 mM NaCl, 10 mM sodium phosphate, 1.0 mM EDTA, 25°C).

[0147] 3. Effects of RNA sequences The structural selectivity of Probe 2 for various RNAs was evaluated according to "4. Effect of RNA sequence" in Example 1. To evaluate the structural selectivity of Probe 2 at pH 7.0, we observed the fluorescence response of Probe 2 to the various RNAs shown in Figure 3. The fluorescence spectrum area (520-700 nm) normalized with the response to the target RNA set to 1 is summarized in a bar graph. Probe 2 gave a selective fluorescence response to the target RNA, and was shown to have higher selectivity for natural mutant RNA than Probe 1 (Figure 16).

[0148] 4. Calculation of dissociation constants from fluorescence experiments The binding strength of probe 2 to the target RNA was quantitatively evaluated by a fluorescence titration experiment. The reagents used other than probe 2 and their concentrations, and the conditions of the fluorometer were as described in [3. Fluorescence measurement] of Example 1. As shown in Figure 17A and B, the concentration dependence of the obtained fluorescence intensity was analyzed using a 1:1 binding fitting equation, and it was found that probe 2 had a dissociation constant K d The binding was found to be 185 ± 31 nM (n = 3). This was weaker than that of probe 1, but the dissociation constant (K d = 50.5 μM).

[0149] Example 3 1. Probe 3 Design Probe 3 was synthesized, which is different from the tFIT-DPQ probe (probe 1) synthesized in Example 1. The structural formula and chemical formula of probe 3 are shown in Figures 18A and 18B, respectively. The structure of probe 3 differs from that of probe 1 in that the first base C next to the NH2 terminus of the PNA chain is deleted, and the amino acid that binds to DPQ in probe 1 is 2,3-diaminopropionic acid (Dap). Probe 3 was synthesized according to "2. Probe design" in Example 1, except that the PNA sequence and a portion of the amino acid linker were changed from those of probe 1.

[0150] 2. Fluorescence Measurement The interaction between probe 3 and RNA was analyzed according to [3. Fluorescence measurement] of Example 1. The reagents used other than probe 3 and their concentrations, and the conditions of the fluorometer were as described in [3. Fluorescence measurement] of Example 1. As shown in Figure 19, when probe 3 was used, no fluorescence was emitted in the absence of RNA, but light-up type fluorescence was observed, which indicates a fluorescent response in the presence of target RNA. This light-up response was observed even under neutral conditions of pH 7.0 (buffer containing 100 mM NaCl, 10 mM sodium phosphate, 1.0 mM EDTA, 25°C).

[0151] 3. Effects of RNA sequences The structural selectivity of Probe 3 for various RNAs was evaluated according to "4. Effect of RNA sequence" in Example 1. To evaluate the structural selectivity of Probe 3 at pH 7.0, we observed the fluorescence response of Probe 3 to the various RNAs shown in Figure 3. The fluorescence spectrum area (520-700 nm) normalized with the response to the target RNA set to 1 is summarized in a bar graph. Probe 3 gave a selective fluorescence response to the target RNA, and was shown to have extremely higher selectivity for No Loop RNA than Probe 1 (Figure 20).

[0152] 4. Calculation of dissociation constants from fluorescence experiments The binding strength of probe 3 to the target RNA was quantitatively evaluated by a fluorescence titration experiment. The reagents used other than probe 2 and their concentrations, and the conditions of the fluorometer were as described in Example 1 [3. Fluorescence measurement]. As shown in Figures 21A and 21B, the concentration dependence of the obtained fluorescence intensity was analyzed using a 1:1 binding fitting equation, and it was found that probe 3 had a dissociation constant K d = 150 ± 71 nM (n = 3). This is weaker than that of probe 1, but the dissociation constant (K d = 50.5 μM).

[0153] 5. Evaluation of endonuclease activity inhibition using viral polymerase According to "7. Evaluation of ability to inhibit endonuclease activity using viral polymerase" in Example 1, in this experiment, the ability of the probe to inhibit transcription in vitro was evaluated. As shown in FIG. 22, it was suggested that probe 3 had a higher ability to inhibit mRNA transcription than probe 1.

[0154] Example 4 1. Probe 4 Design Probe 4 was synthesized, which is different from the tFIT-DPQ probe (probe 1) synthesized in Example 1. The structural formula and chemical formula of probe 4 are shown in Figures 23A and 23B, respectively. The structure of probe 4 differs from that of probe 1 in that the first base C next to the NH2 terminus of the PNA chain is deleted and the thiazole orange (TO) spacer is two carbons longer (Figure 23C). Probe 4 was synthesized according to "2. Probe design" in Example 1, except that the PNA sequence was changed from that of probe 1.

[0155] 2. Fluorescence Measurement The interaction between probe 4 and RNA was analyzed according to [3. Fluorescence measurement] of Example 1. The reagents used other than probe 4 and their concentrations, and the conditions of the fluorometer were as described in [3. Fluorescence measurement] of Example 1. As shown in Figure 24, when probe 4 was used, no fluorescence was emitted in the absence of RNA, but light-up type fluorescence was observed, which indicates a fluorescent response in the presence of target RNA. This light-up response was observed even under neutral conditions of pH 7.0 (buffer containing 100 mM NaCl, 10 mM sodium phosphate, 1.0 mM EDTA, 25°C).

[0156] 3. Calculation of dissociation constants from fluorescence experiments The binding strength of probe 4 to the target RNA was quantitatively evaluated by a fluorescence titration experiment. The reagents used other than probe 4 and their concentrations, and the conditions of the fluorometer were as described in [3. Fluorescence measurement] of Example 1. As shown in Figures 25A and 25B, the concentration dependence of the obtained fluorescence intensity was analyzed using a 1:1 binding fitting equation, and it was found that probe 2 binds to the target RNA with a dissociation constant of 52.6 ± 16 nM (n = 3). This is weaker than probe 1, but is close to the dissociation constant (K d = 50.5 μM).

Claims

1. A complex that inhibits transcription or replication of a viral RNA, the complex targeting a promoter region including a first nucleic acid sequence extending from the 5' end to the 3' end, a second nucleic acid sequence located on the 3' end side of the first nucleic acid sequence and forming a double strand with the first nucleic acid sequence, and an internal loop formed between the double strand, a PNA strand having a sequence that is completely complementary to a target nucleic acid sequence from the 5' end to the 8th or 9th base of the first nucleic acid sequence of the RNA, or a sequence that is complementary except for one base, and having 8, 9 or 10 bases, and the 3rd or 4th PNA from the N-terminus of the PNA strand is substituted with methinecyanine; 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ), a linker connecting the PNA strand and the DPQ; A complex comprising:

2. The conjugate of claim 1, wherein the methine cyanine is selected from thiazole orange (TO), quinoline blue (QB), and thiazole red (TR).

3. The base sequence of the PNA strand is The complex according to claim 1 or 2, which has an N-terminus -NTCNTCTTT-C-terminus (wherein the first N is no base or C, and the fourth N is G, C, A, T or methinecyanine).

4. The complex according to any one of claims 1 to 3, wherein the RNA is influenza A virus RNA.

5. A viral RNA transcription inhibitor comprising the complex according to any one of claims 1 to 4.

6. An inhibitor of viral RNA replication, comprising the complex according to any one of claims 1 to 4.

7. A pharmaceutical composition for inhibiting the transcription or replication of viral RNA, comprising the complex according to any one of claims 1 to 4.

8. A pharmaceutical composition for preventing or treating a viral infection, comprising the complex according to any one of claims 1 to 4.

9. 1. A method for inhibiting RNA transcription or replication in vitro or in a non-human mammalian subject, comprising: contacting RNA with a complex according to any one of claims 1 to 4, the RNA is a viral RNA that targets a promoter region extending from the 5' end to the 3' end and that comprises a first nucleic acid sequence, a second nucleic acid sequence that is located on the 3' end side of the first nucleic acid sequence and forms a double strand with the first nucleic acid sequence, and an internal loop formed between the double strand; The complex is a PNA strand having a sequence that is completely complementary to a target nucleic acid sequence from the 5' end to the 8th or 9th base of the first nucleic acid sequence of the RNA, or a sequence that is complementary except for one base, and having 8, 9 or 10 bases, and the 3rd or 4th PNA from the 5' end of the PNA strand is substituted with methinecyanine; 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ), a linker connecting the PNA strand and the DPQ; A method comprising:

10. A method for screening a compound capable of inhibiting RNA replication or transcription, comprising the steps of: A step of previously binding RNA to the complex according to any one of claims 1 to 4 to form a conjugate; contacting the conjugate with a test compound; and measuring each signal value obtained from the bound body before and after the contact; A screening method comprising:

11. The method of claim 10, wherein the test compound is determined to be a hit compound if the signal value after contacting the test compound is lower than the signal value before contacting the test compound.

12. A kit for evaluating the ability of a test compound to inhibit RNA replication or transcription, comprising: A kit comprising the complex according to any one of claims 1 to 4.

13. 1. A kit for assessing viral infection, comprising: A kit comprising the complex according to any one of claims 1 to 4.

14. An in vitro test for assessing a viral infection, comprising the complex according to any one of claims 1 to 4.

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