Use of pi4k inhibitors in the preparation of a medicament for combating coronavirus

Abstract

Use of phenylarsine oxide and its derivatives in preventing or treating coronavirus diseases, and use of PI4KIIIα specific inhibitors in preventing or treating coronavirus diseases are provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of PI4KIIIα specific inhibitors in the preparation of a medicament for preventing or treating a coronavirus disease. BACKGROUND

[0002] Coronaviruses have been widely concerned due to their high infectivity. For example, the mortality rates of two highly pathogenic coronaviruses, Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle east respiratory syndrome coronavirus (MERS-CoV), are as high as 10% and 36%, respectively; Human coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and HCoV-HKU8) cause 15%-30% of upper respiratory tract infections in humans every year, and the incidence is higher in the population of newborns, the elderly and individuals with underlying diseases, thus coronaviruses have been paid great attention.

[0003] It is generally believed that the entry of coronaviruses into host cells is through a pH-dependent pathway, i.e. after the virus is adsorbed on the cell surface, the cell membrane invaginates, the virus is internalized, and the viral RNA is released into the cytoplasm. Thereafter, the viral RdRp is first synthesized, which is a polymerase that recognizes the positive-strand RNA of the coronavirus genome and synthesizes negative-strand RNA as a template, and then synthesizes subgenomic small positive-strand RNA and positive-strand genomic RNA as a template. In the cytoplasm, ribosomes translate the subgenomic small positive-strand RNA as a template to generate precursor proteins. Thereafter, protein N binds to newly synthesized genomic RNA, and under the participation of protein M, reaches the endoplasmic reticulum, integrates with protein S and is released from the endoplasmic reticulum membrane. At the same time, proteases cut the progeny virus precursor proteins into mature proteins. Thereafter, the progeny virus is transferred from the Golgi apparatus to the cell membrane and released to the outside of the cell (Masters et al., The molecular biology of coronaviruses. Adv Virus Res. 2006. Vol 66: p. 193-292) to start a new round of infection of cells, replication in cells and damage to cells, thereby affecting the normal function of the body.

[0004] Recently, a new coronavirus (SARS-CoV-2), also known as the novel coronavirus, has received much attention from various countries due to its high infectivity, which causes Novel coronavirus pneumonia (NCP) with an expected transmission base RO value between 2-6. Like other coronaviruses, SARS-CoV-2 is an enveloped virus. The genetic material of SARS-CoV-2 is a positive single-stranded RNA virus, and its gene sequence belongs to the same lineage as SARS and MERS viruses.

[0005] According to current speculation, SARS-CoV-2, like other coronaviruses, stimulates the innate immune system of patients, causing a large amount of cytokine release in the body, resulting in a cytokine storm and acute inflammatory response. This can cause systemic blood vessels to be more fragile, leading to acute respiratory distress syndrome and multiple organ failure (Huang et al., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020: p1-10; Kikkert et al., Innate Immune Evasion by Human Respiratory RNA Viruses. Journal of Innate Immunity. 2020, 12(1)). Recent evidence shows that SARS-CoV-2 appears in the cerebrospinal fluid of patients with Novel coronavirus pneumonia, which is consistent with the symptoms of neck resistance, sudden onset of consciousness, and coma in patients, indicating that in addition to the lungs, heart, blood, liver, and other multiple organs, the virus can also attack the nervous system.

[0006] Similar to SARS-CoV, the SARS-CoV-2 protein S can recognize and bind to the host surface receptor angiotensin-converting enzyme 2 (ACE2), allowing the virus to adsorb to the cell surface. The binding force of ACE2 to the SARS-CoV-2 virus is believed to be stronger than that to SARS-CoV (Xu et al., Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. SCIENCE CHINA Life Sciences. 2020, Vol 63, p. 457-460). There is currently no report of a drug that has a significant therapeutic effect on SARS-CoV-2 infection.

[0007] Phenylarsine oxide (PAO) is a known biological inhibitor,

[0008]

[0009] The arsenic atom in the molecule has a high affinity for the sulfur atom of the thiol group in the biological molecule. There is currently no report that phenylarsine oxide and its derivatives can be used to treat coronavirus diseases including the novel coronavirus. SUMMARY

[0010] In one aspect, the present application provides use of a PI4KIIIa specific inhibitor in the manufacture of a medicament for preventing or treating a coronavirus disease.

[0011] In some embodiments, the PI4KIIIa specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, or an antisense nucleic acid.

[0012] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0013] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0014] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

[0015] In some embodiments, the RNAi molecule has a length of 18-100 bases.

[0016] In some embodiments, the RNAi molecule is modified to enhance its stability.

[0017] In some embodiments, the PI4KIIIa specific inhibitor is a small molecule compound.

[0018] In some embodiments, the small molecule compound is an arsenoxide or a derivative thereof, G1 and analogs thereof, A1 and analogs thereof, or simeprevir and analogs thereof.

[0019] In some embodiments, the arsenoxide and derivatives thereof have a structure represented by Formula (I) or a pharmaceutically acceptable salt thereof,

[0020]

[0021] wherein each R1is independently selected from (a) H, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkylene-NH2, C 1-6 alkylene-NH-C(O)H, -As(O), -N=NH, N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, -NH-S(O)2H, -C(O)OH, -OC(O)H, -SH, -S(O)2H, -S(O)2-NH2, or heterocyclyl, and optionally substituted with R2or R3, wherein each of R2and R3is independently selected from amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, N-(C 1-6 alkyl)amino, N-(6-12 membered aryl)amino, N,N-(C 1-6 alkyl)2amino, C 3-6 cycloalkyl, 6-12 membered aryl, or 3-12 membered heterocyclyl, and optionally substituted with one or more halogen, nitro, cyano, hydroxyl, amino, carbamoyl, -NH-C(O)-R5, -C(O)OR4, 6-12 membered aryl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, 3-6 membered heterocyclyl, C 3-6 cycloalkyl, or Bn-O-, and R4is C 1-6The alkyl group, and optionally with one or more halogens, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 aryl groups, C 1-6 Alkyl, C 2-6 alkynyl group, C 2-6 alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted, R5 is selected from H, C 1-6 Alkyl, C 2-6 alkynyl group, C 2-6 alkenyl, C 1-6 Alkoxy or C 1-6 Halogenated alkyl groups, and / or

[0022] (b) R1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic, or heterocyclic group, and optionally is radically substituted with one or more halogens, nitro, cyano, hydroxyl, amino, carbamoyl, 6-12 membered aromatic groups, C 1-6 Alkyl, C 2-6 alkynyl group, C 2-6 alkenyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, 3-6 membered heterocyclic group, C 3-6 Cycloalkyl or Bn-O-substituted,

[0023] Where n is an integer between 0 and 5.

[0024] In some implementations, n is an integer from 0 to 2, and each of R1 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, -As(O), N-(C) 1-6 Alkyl)amino, N,N-(C) 1-6 Alkyl)2amino, -NH-C(O)H or -NH-S(O)2H, and optionally substituted with said R2 or R3.

[0025] In some implementations, n is an integer from 0 to 2, and each of R1 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 The alkyl halogroup is -As(O), -NH-C(O)H or -NH-S(O)2H, and optionally substituted with said R2 or R3.

[0026] In some embodiments, n is 1 or 2, and each of R1 is independently selected from H, halogen, amino, C. 1-6alkyl, C 1-6 haloalkyl, -NH-C(O)R2or -NH-S(O)2R3, wherein R2is C 1-6 alkyl, C 1-6 alkyl, C 1-6 haloalkyl, -NH-C(O)R2or -NH-S(O)2R3, wherein R2is C

[0027] In some embodiments, the R1is located at the ortho and / or para position of the -As(O) group.

[0028] In some embodiments, n is 0.

[0029] In some embodiments, the small molecule compound is selected from the group consisting of:

[0030]

[0031]

[0032]

[0033]

[0034] In some embodiments, the subject is a human or a mammal.

[0035] In some embodiments, the coronavirus is a novel coronavirus.

[0036] In some embodiments, the coronavirus is infectious bronchitis virus of chicken, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine hemagglutinating encephalomyelitis virus, porcine delta coronavirus, canine respiratory coronavirus, mouse hepatitis virus, feline coronavirus, human coronavirus, severe acute respiratory syndrome virus, Middle East respiratory syndrome virus.

[0037] In some embodiments, further comprising administering to the subject in need thereof a second agent.

[0038] In some embodiments, the second agent is an agent for treating a coronavirus disease.

[0039] In some embodiments, the PI4KIIIa specific inhibitor is administered before, after or concurrently with the second agent.

[0040] In another aspect, the present application provides a method of screening a drug for preventing or treating a coronavirus disease, comprising contacting a candidate drug with a PI4KIIIa protein or nucleic acid or PI4KIIIa and detecting whether the candidate drug is capable of inhibiting the formation or activity of PI4KIIIa.

[0041] In yet another aspect, the present application provides use of a compound of formula (I) in the manufacture of a medicament for the prevention or treatment of a disease of the coronavirus class:

[0042]

[0043] wherein each R1is independently selected from (a) H, halogen, nitro, cyano, hydroxy, amino, carbamoyl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkylene-NH2, C 1-6 alkylene-NH-C(O)H, -As(O), -N=NH, N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, -NH-S(O)2H, -C(O)OH, -OC(O)H, -SH, -S(O)2H, -S(O)2-NH2, or heterocyclyl, and optionally substituted with R2or R3, wherein each of R2and R3is independently selected from amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, N-(C 1-6 alkyl)amino, N-(6-12 membered aryl)amino, N,N-(C 1-6 alkyl)2amino, C 3-6 cycloalkyl, 6-12 membered aryl, or 3-12 membered heterocyclyl, and optionally substituted with one or more halogen, nitro, cyano, hydroxy, amino, carbamoyl, -NH-C(O)-R5, -C(O)OR4, 6-12 membered aryl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, 3-6 membered heterocyclyl, C 3-6 cycloalkyl, or Bn-O-, and R4is C 1-6 alkyl, and optionally substituted with one or more halogen, nitro, cyano, hydroxy, amino, carbamoyl, 6-12 membered aryl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, 3-6 membered heterocyclyl, C 3-6cycloalkyl or Bn-O-substituted, R5is selected from H, C 1-6 alkyl, C 2-6 alkynyl, C 2-6 alkenyl, C 1-6 alkoxy, or C 1-6 haloalkyl, and / or

[0044] (b) R1on two adjacent carbon atoms form a 5-12 membered cycloalkyl, aryl or heterocyclyl group, and optionally substituted by one or more halogen, nitro, cyano, hydroxy, amino, carbamoyl, 6-12 membered aryl, C 1-6 alkyl, C 2-6 alkynyl, C 2-6 alkenyl, C 1-6 alkoxy, C 1-6 haloalkyl, 3-6 membered heterocyclyl, C 3-6 cycloalkyl or Bn-O-substituted,

[0045] wherein n is an integer from 0 to 5.

[0046] In some embodiments, the compound is phenarsazine oxide. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 shows the toxic effect of different concentrations of phenarsazine oxide on Vero cells. Figure 1A shows the toxic effect of PI01 small molecule drug at a concentration of 200 nM on Vero cells, Figure 1B shows the toxic effect of PI01 small molecule drug at a concentration of 200 nM on Vero cells, Figure 1C shows the toxic effect of different concentrations of Remdesivir on Vero cells, Figure 1D shows the toxic effect of different concentrations of Chloroquine phosphate on Vero cells.

[0048] Figure 2 shows the effect of different concentrations of phenarsazine oxide on the inhibition of virus in vitro. Figure 2A shows the effect of different concentrations of PI01 drug on the inhibition of SARS-CoV-2 in Vero cells, Figure 2B shows the effect of different concentrations of PI01 drug on the inhibition of SARS-CoV-2 in Vero cells, Figure 2C shows the effect of different concentrations of Remdesivir on the inhibition of SARS-CoV-2 in Vero cells, Figure 2D shows the effect of different concentrations of Chloroquine phosphate on the inhibition of SARS-CoV-2 in Vero cells.

[0049] Figure 3 shows the activity of different agents against human coronavirus HCoV-229E in MRC5 cells.Figure 3A showing the results of the first round, Figure 3B showing the results of the second round. DETAILED DESCRIPTION

[0050] The present application is described in detail below according to embodiments, with reference to the attached drawings. The above and further aspects of the present application will become apparent from the following detailed description, particularly when reviewed in conjunction with the drawings. The application is not limited to the described embodiments.

[0051] PI4KIIIa specific inhibitors

[0052] The term "PI4KIIIa specific inhibitor" as used herein refers to any substance that is capable of specifically reducing, decreasing, eliminating the transcription or translation of the PI4KIIIa gene and / or the activity of the PI4KIIIa protein. In some embodiments, the PI4KIIIa specific inhibitor is capable of reducing the activity of PI4KIIIa by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more. In this context, "activity" when used in conjunction with increasing or decreasing refers to the detected functional activity, which can be manifested as a change in amount or a change in functional activity without a change in amount.

[0053] In this context, the activity of PI4KIIIa refers to the activity of the PI4KIIIa protein to phosphorylate a phosphoinositide (PI) at a specific position (e.g., to 4-phospho phosphatidylinositol (PI4P)). The binding constant of the PI4KIIIa specific inhibitor to the PI4KIIIa protein is at least 2-fold or more than the binding constant to other non-specific binding proteins. In some embodiments, the PI4KIIIa specific inhibitor is capable of preferentially recognizing the PI4KIIIa protein in a complex mixture, including a mixture with other PI4K isoform proteins.

[0054] In some embodiments, the PI4KIIIa specific inhibitor inhibits PI4KIII at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold or 10,000-fold stronger than other PI4K protein isoforms (e.g., PI4K proteins including PI4KIIa or PI4KIIb). For example, in some embodiments, the PI4KIIIa specific inhibitor does not substantially inhibit PI4KII (e.g., PI4KIIa or PI4KIIb), e.g., its IC50 is greater than or equal to 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM or 500 μM. 50 greater than or equal to 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM or 500 μM.

[0055] In some embodiments, the PI4KIIIa specific inhibitor inhibits PI4KIIIa at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 10000-fold stronger than other isoforms of PI4KIII (e.g., PI4KIII beta).

[0056] In some embodiments, the PI4KIIIa specific inhibitor has an IC50 for PI4KIIIa that is less than or equal to 100 μΜ, 80 μΜ, 50 μΜ, 30 μΜ, 20 μΜ, 10 μΜ, 5 μΜ, 3 μΜ, 2 μΜ, 1 μΜ, 0.5 μΜ, 0.2 μΜ, 0.1 μΜ, 0.05 μΜ, 0.02 μΜ, 0.01 μΜ, 0.005 μΜ, 0.002 μΜ, or 0.001 μΜ. In some embodiments, the PI4KIIIa specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, or an antisense nucleic acid. 50 In some embodiments, the PI4KIIIa specific inhibitor has an IC50 for PI4KIIIa that is less than or equal to 100 μΜ, 80 μΜ, 50 μΜ, 30 μΜ, 20 μΜ, 10 μΜ, 5 μΜ, 3 μΜ, 2 μΜ, 1 μΜ, 0.5 μΜ, 0.2 μΜ, 0.1 μΜ, 0.05 μΜ, 0.02 μΜ, 0.01 μΜ, 0.005 μΜ, 0.002 μΜ, or 0.001 μΜ. In some embodiments, the PI4KIIIa specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, or an antisense nucleic acid.

[0057] In some embodiments, the PI4KIIIa specific inhibitor is an antibody.

[0058] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, or antibody that can bind to a particular antigen. The term "antibody" herein is intended to broadly encompass conventional four-chain antibodies as well as less conventional antibodies that do not have four chains (e.g., antibodies that naturally lack light chains).

[0059] A conventional full antibody is a heterotetramer composed of two heavy (H) chains and two light (L) chains. A heavy chain of a mammal can be classified as alpha, delta, epsilon, gamma, and mu, each of which can comprise a variable region (VH) and a first, second, and third constant regions (CH1, CH2, CH3, respectively); a light chain of a mammal can be classified as lambda or kappa, each of which can comprise a variable region (VL) and a constant region. A conventional antibody is in the shape of a "Y", with the neck of the "Y" shape comprising the second and third constant regions of the two heavy chains, which are joined by disulfide bonds. Each arm of the "Y" shape comprises the variable region and the first constant region of one of the heavy chains, which are combined with the variable region and the constant region of one of the light chains. The variable regions of the light and heavy chains determine antigen binding. Each variable region contains three hypervariable regions, termed complementarity determining regions (CDRs) (the CDRs of a light chain comprise LCDR1, LCDR2, LCDR3, and the CDRs of a heavy chain comprise HCDR1, HCDR2, HCDR3). The three CDRs are separated by relatively non-variable portions known as framework regions (FRs), which form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.

[0060] In some embodiments of the application, the antibody is a full-length antibody or an antigen-binding fragment.

[0061] The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from a portion of an antibody that contains one or more CDRs but does not have the structure of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single domain antibodies, and nanobodies. An antigen-binding fragment can bind to the same antigen as the parent antibody.

[0062] The "Fab" fragment of an antibody refers to that portion of an antibody that is bonded by a disulfide bond to a portion of a light chain (including variable and constant regions) and a variable region and first constant region of a heavy chain.

[0063] The "Fab'" fragment refers to a Fab fragment that includes a portion of the hinge region.

[0064] The "F(ab')2" fragment refers to a dimer of Fab'.

[0065] The "Fv" fragment of an antibody is composed of the variable regions of a light chain and those of a heavy chain.

[0066] A "single-chain antibody molecule" or "scFv" refers to an engineered antibody in which a light chain variable region is directly or indirectly linked to a heavy chain variable region by a peptide chain. See, e.g., Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988).

[0067] A "scFv dimer" refers to a polymer formed from two scFv.

[0068] A "camelized single domain antibody" (also referred to as a "heavy-chain antibody" or "HCAb") refers to an antibody that contains two heavy chain variable regions and no light chain. Heavy-chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas). Despite the absence of a light chain, camelized antibodies have full functionality for antigen binding.

[0069] A "nanobody" is composed of a heavy chain variable region from a heavy-chain antibody and two constant regions, CH2 and CH3.

[0070] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0071] In some embodiments, the antibody is a murine antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0072] The term "fully human" as used herein when referring to an antibody or antigen binding fragment means that the amino acid sequence of the antibody or antigen binding fragment corresponds to the amino acid sequence of an antibody produced by a human or human immune cell, or derived from an antibody of non-human origin, such as a transgenic non-human animal that utilizes a human antibody repertoire, or other sequence encoding a human antibody.

[0073] The term "humanized" as used herein when referring to an antibody or antigen binding fragment means an antibody or antigen binding fragment that includes CDRs derived from a non-human animal, FR regions derived from a human, and a constant region derived from a human, when applicable. Because humanized antibodies or antigen binding fragments have lower immunogenicity, they can be used as therapeutic agents in humans in certain embodiments. In certain embodiments, the non-human animal is a mammal (e.g., a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster). In certain embodiments, the humanized antibody or antigen binding fragment consists essentially of human sequences except for the CDR sequences, which are of non-human origin.

[0074] The term "chimeric" as used herein when referring to an antibody or antigen binding fragment means an antibody or antigen binding fragment that has a portion of the heavy and / or light chain derived from one species and the remainder of the heavy and / or light chain derived from different species. In some embodiments, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse or a rabbit).

[0075] In some embodiments, the antibodies described herein are monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0076] In some embodiments, the antibodies described herein can be further labeled.

[0077] In some embodiments, the PI4KIIIa specific inhibitor is an RNAi molecule.

[0078] The term "RNAi molecule" as used herein refers to an RNA or an analog thereof that has sufficient sequence complementarity to a target RNA to direct RNA interference. In some embodiments, DNA that can be used to generate the RNA is also included. RNA interference (RNAi) refers to a sequence-specific or selective process by which a target molecule (e.g., a target gene, protein, or RNA) is down-regulated.

[0079] In some embodiments, the RNAi molecule is capable of reducing the expression of PI4KIIIa, e.g., knocking down the PI4KA gene.

[0080] In some embodiments, the RNAi molecule is 18-100 bases in length.

[0081] In some embodiments, the RNAi molecule is modified to enhance its stability.

[0082] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a micro RNA (miRNA).

[0083] The term "small interfering RNA (siRNA)", as used herein, refers to an RNA molecule, preferably a double-stranded molecule, having a length of about 10-50 nucleotides, preferably about 15-25 nucleotides, more preferably about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, the strands optionally having overhanging ends comprising, for example, 1, 2, or 3 overhanging nucleotides (or nucleotide analogs) that are capable of directing or mediating degradation of RNA.

[0084] The term "short hairpin RNA (shRNA)", as used herein, refers to an RNA molecule having a stem-loop structure, comprising a first region and a second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient to have base pairs occurring between the regions, the first region and the second region being connected by a loop region, the loop resulting from the absence of base pairing between nucleotides (or nucleotide analogs) in the loop region.

[0085] The term "microRNA (or miRNA)" as used herein refers to a short, naturally occurring, non-coding, single-stranded RNA molecule of about 16-26 nucleotides (nt) in length (e.g., about 16-29 nt, 19-22 nt, 20-25 nt, or 21-23 nt) that is generally involved in the regulation of gene expression in vivo. In eukaryotic cells, miRNA genes are transcribed by DNA transcriptase II into a "primary product" (pri-miRNA), which is quickly processed by a ribonuclease III (Drosha) into a miRNA "precursor" (pre-miRNA), which is transported from the nucleus to the cytoplasm, where it is recognized and cleaved by another ribonuclease III (Dicer) into a mature miRNA. The mature miRNA molecule is complementary to one or more mRNA portions and regulates the expression of a protein. Known sequences of miRNAs are available in public databases, such as the miRBase database (www.mirbase.org), which provides information including miRNA sequence information, functional annotations, and predicted gene targets. In the present application, miRNA also includes RNA molecules expressed in cells by artificially synthesized plasmids, which have similar structure and function to natural miRNAs, and can target corresponding mRNAs like natural miRNAs to hinder their translation into proteins.

[0086] In some embodiments, the PI4KIIIa specific inhibitor is an antisense nucleic acid.

[0087] The term "antisense nucleic acid" as used herein includes nucleotides that are completely complementary to a target sequence as well as those nucleotides having one or more nucleotide mismatches, as long as the antisense nucleic acid is capable of specifically hybridizing to the target sequence. For example, an antisense nucleic acid herein includes a polynucleotide having at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more homology over a length of at least 15 contiguous nucleotides. As a result of the formation of a hybrid, target gene transcription and / or target mRNA translation is reduced or blocked.

[0088] In some embodiments, the PI4KIIIa inhibitor is a small molecule compound.

[0089] The term "small molecule compound" as used herein refers to an organic compound having a molecular weight of less than 3000, 2500, 2000, 1500, 1000, or 500 daltons, which can be natural or chemically synthesized.

[0090] wherein the small molecule compound has a structure represented by Formula (I) or a pharmaceutically acceptable salt thereof,

[0091]

[0092] wherein each R1is independently selected from the group consisting of (a) H, halo, nitro, cyano, hydroxy, amino, carbamoyl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkylene-NH2, C 1-6 alkylene-NH-C(O)H, -As(O), -N=NH, N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, -NH-S(O)2H, -C(O)OH, -OC(O)H, -SH, -S(O)2H, -S(O)2-NH2, or heterocyclyl, and optionally substituted with R2or R3, wherein each of R2and R3is independently selected from the group consisting of amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, N-(C 1-6 alkyl)amino, N-(6-12 membered aryl)amino, N,N-(C 1-6 alkyl)2amino, C 3-6 cycloalkyl, 6-12 membered aryl, or 3-12 membered heterocyclyl, and optionally substituted with one or more halo, nitro, cyano, hydroxy, amino, carbamoyl, -NH-C(O)-R5, -C(O)OR4, 6-12 membered aryl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, 3-6 membered heterocyclyl, C 3-6 cycloalkyl, or Bn-O-, and R4is C 1-6 alkyl, and optionally substituted with one or more halo, nitro, cyano, hydroxy, amino, carbamoyl, 6-12 membered aryl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, 3-6 membered heterocyclyl, C 3-6 cycloalkyl, or Bn-O-, and R5is selected from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, or C 1-6haloalkyl, and / or

[0093] (b) two R1on adjacent carbon atoms form a 5-12 membered cycloalkyl, aryl, or heterocyclyl group, and are optionally substituted with one or more halogen, nitro, cyano, hydroxy, amino, carbamoyl, 6-12 membered aryl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, 3-6 membered heterocyclyl, C 3-6 cycloalkyl, or Bn-O-, and

[0094] wherein n is an integer from 0 to 5.

[0095] In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0 or 1.

[0096] In some embodiments, n is an integer from 0 to 2, each R1is independently selected from H, halogen, nitro, cyano, hydroxy, amino, carbamoyl, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, -As(O), N-(C 1-6 alkyl)amino, N,N-(C 1-6 alkyl)2amino, -NH-C(O)H, or -NH-S(O)2H, and is optionally substituted with R2or R3.

[0097] In some embodiments, n is an integer from 0 to 2, each R1is independently selected from H, halogen, nitro, cyano, hydroxy, amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, -As(O), -NH-C(O)H, or -NH-S(O)2H, and is optionally substituted with R2or R3.

[0098] In some embodiments, n is 1 or 2, each R1is independently selected from H, halogen, amino, C 1-6 alkoxy, C 1-6 haloalkyl, -NH-C(O)R2, or -NH-S(O)2R3, wherein R2is C 1-6 alkyl, optionally substituted with one 6-12 membered aryl, and R3is 6-12 membered aryl, optionally substituted with one halogen, C 1-6 alkoxy, or C 1-6 haloalkyl.

[0099] In some embodiments, said R1is located at the ortho and / or para position of the -As(O) group.

[0100] In some embodiments, said R1is H.

[0101] The term "substituted", as used herein when referring to a chemical group, means that one or more hydrogen atoms of the chemical group are removed and replaced with a substituent.

[0102] The term "substituent", as used herein, has its ordinary meaning in the art, referring to a chemical moiety covalently attached to or, where appropriate, fused to a parent group.

[0103] The term "C n -C m " denotes a range of the number of carbon atoms, wherein n and m are integers and the range of the number of carbon atoms includes the endpoints (i.e., n and m) and each integer point in between. For example, C1-C6denotes a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms.

[0104] The term "alkyl", whether preceeded by the term "C or used alone, refers to a saturated hydrocarbon group, which can be straight chain or branched chain. The term "C n -C m alkyl" refers to an alkyl group having n to m carbon atoms. In certain embodiments, the alkyl group contains 1 to 12, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of alkyl groups include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, 2-methyl-l-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like.

[0105] The term "alkenyl", whether preceeded by the term "C or used alone, refers to an unsaturated hydrocarbon group, which can be straight chain or branched chain, having at least one carbon-carbon double bond. In certain embodiments, the alkenyl group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. In certain embodiments, the alkenyl group can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, chemical groups such as ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

[0106] The term "alkynyl," as used herein, whether used alone or as part of another term, refers to an unsaturated alkynyl group that can be straight-chain or branched and has at least one carbon-carbon triple bond. In certain embodiments, the alkynyl group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. In certain embodiments, the alkynyl group can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, chemical groups such as ethynyl, propynyl, butynyl, and the like.

[0107] The term "cycloalkyl," as used herein, refers to a cyclic ring system alkyl group consisting of at least 3 atoms. The term "n-m membered cycloalkyl" refers to a cycloalkyl group having n to m members forming a ring. In addition, the ring can also have one or more double bonds, but not a fully conjugated system. In certain embodiments, the cycloalkyl group has 3 to 8, 3 to 6, or 4 to 6 carbon atoms forming a ring. Examples of cycloalkyl groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentyl, and the like.

[0108] The term "heterocyclyl," as used herein, refers to a ring group in which at least one of the atoms in the ring system is a heteroatom and the remaining ring atoms are carbon atoms. The term "n-m membered heterocyclyl" refers to a heterocyclyl group having n to m members forming a ring. The term "heterocyclyl," as used herein, includes both heteroaryl and heterocycloalkyl groups. In addition, the ring can also have one or more double bonds. In certain embodiments, the heterocyclyl group is a saturated heterocycloalkyl group. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like.

[0109] The term "heterocycloalkyl," as used herein, refers to a cycloalkyl group in which at least one of the atoms in the ring system is a heteroatom and the remaining ring atoms are carbon atoms. The term "n-m membered heterocycloalkyl" refers to a heterocycloalkyl group having n to m members forming a ring. In addition, the ring can also have one or more double bonds, but not a fully conjugated system. In certain embodiments, the heterocycloalkyl group is a saturated heterocycloalkyl group. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like. In certain embodiments, the heterocycloalkyl group has 3 to 8, 3 to 6, or 4 to 6 carbon atoms forming a ring. Examples of heterocycloalkyl groups include, but are not limited to, azetidine, aziridine, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazine, and the like.

[0110] The term "aryl" or "aromatic," as used herein, whether used alone or as part of another term, refers to a mono- or poly-carbocyclic ring system group having alternating single and double bonds between the carbon atoms that form the ring. The term "C n -C m"Aryl" refers to an aryl radical having n to m ring-forming carbon atoms. In certain embodiments, the aryl ring system has 6 to 12, 6 to 10, or 6 to 8 carbon atoms in one or more rings. In certain embodiments, the aryl ring system has 2 or more rings that are fused together. Examples of aryl groups include, but are not limited to, chemical groups such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.

[0111] The term "heteroaryl" as used herein refers to an aryl group in which at least one ring atom in the aromatic ring is a heteroatom and the remaining ring atoms are carbon atoms. The term "n-m membered heteroaryl" refers to a heteroaryl group having n to m ring-forming members. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like. In certain embodiments, the heteroaryl group can have 5 to 10, 5 to 8, or 5 to 6 ring-forming members. In certain embodiments, the heteroaryl group is a 5- or 6-membered heteroaryl group. Examples of heteroaryl groups include, but are not limited to, furanyl, thienyl, pyridyl, quinolinyl, pyrrolyl, N-lower alkyl pyrrolyl, pyridyl-N-oxide, pyrimidinyl, pyrazinyl, imidazolyl, indolyl, and the like.

[0112] The term "alkoxy," whether used as a part of another term or used alone, as used herein refers to a group represented by the formula "-O-alkyl." The term "C n -C m "Alkoxy" refers to the alkyl portion of the alkoxy group having n to m carbon atoms. In certain embodiments, the alkyl portion has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkoxy groups include, but are not limited to, chemical groups such as methoxy, ethoxy, propyloxy (e.g., n-propyloxy and isopropyloxy), t-butyloxy, and the like.

[0113] The term "haloalkyl," whether used as a part of another term or used alone, as used herein refers to a group represented by the formula "-alkyl-X," wherein X is a halogen, an atom selected from fluorine, chlorine, bromine, and iodine. The term "C n -C m "Haloalkyl" refers to the alkyl portion of the haloalkyl group having n to m carbon atoms. In certain embodiments, the alkyl portion has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of haloalkyl groups include, but are not limited to, chemical groups such as halomethyl, haloethyl, halopropyl (e.g., n-halopropyl and isohalopropyl), t-halobutyl, and the like.

[0114] The term "n-membered", where n is an integer, as used herein is generally used in connection with a ring system to describe the number of atoms forming a ring in the ring system. For example, piperidinyl is one example of a 6-membered heterocycloalkyl ring, pyrazolyl is one example of a 5-membered heteroaryl ring, pyridinyl is one example of a 6-membered heteroaryl ring and 1,2,3,4-tetrahydro-naphthalene is one example of a 10-membered aryl. The term "n- to m-membered", as used herein is generally used in connection with a ring system to describe the range of members forming a ring in the ring system, where n and m are integers and the range of members forming a ring includes the endpoints (i.e., n and m) and each integer point in between. For example, 3- to 8-membered indicates a range of 3 to 8 members forming a ring, including 3 members, 4 members, 5 members, 6 members, 7 members and 8 members.

[0115] The term "halogen", as used herein, means an atom selected from fluorine, chlorine, bromine, and iodine.

[0116] The term "cyano", as used herein, means a group of the formula "-CN".

[0117] The term "hydroxy", as used herein, means a group of the formula "-OH".

[0118] The term "nitro", as used herein, means a group of the formula "-NO2".

[0119] The term "amino", as used herein, means a group of the formula "-NH2".

[0120] The term "carbamoyl", as used herein, means a group of the formula "-HNCONH2".

[0121] The term "compound", as used herein, is intended to include all stereoisomers (e.g., enantiomers and diastereomers), geometric isomers, tautomers, and isotopologues of the depicted structures.

[0122] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, e.g., enantiomers and diastereomers, are intended to be included. Multiple geometric isomers can also exist, e.g., olefinic, carbon-carbon double bond, etc., and all such stable isomers are contemplated to be included herein. Cis and trans geometric isomers are described herein and can be isolated as mixtures of isomers or as separate isomers.

[0123] The compounds herein also include tautomeric forms. Tautomeric forms result from the interchange of a single bond with an adjacent double bond accompanied by the migration of a proton. Tautomeric forms include isomeric protonation states of the same chemical formula and overall charge. Examples of proton tautomers include the keto-enol pair, the amide-imidic acid pair, the lactam-lactim pair, the enamine-imine pair, and cyclic forms in which a proton can occupy two or more positions of a heterocyclic system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or spatially locked into one form by appropriate substitution.

[0124] In certain embodiments, the small molecule compounds herein can be obtained by organic synthesis. The compounds herein, including salts, esters, hydrates, or solvates thereof, can be prepared using any of the well-known techniques of organic synthesis and can be synthesized according to a variety of possible synthetic routes.

[0125] In some embodiments, the small molecule compounds described herein are compounds having the following structural formula, including one or more of:

[0126]

[0127]

[0128]

[0129] As used herein, the term "phenylarsenic oxide" (PAO) refers to a small molecule compound having the following specific chemical structure:

[0130]

[0131] As used herein, the terms "A1" and "G1" are both small molecule compound inhibitors of the PI4KIIIa protein, having similar structures. The chemical structural formula of A1 is:

[0132]

[0133] 5-(2-amino-1-(4-(4-morpholinyl)phenyl)-1H-benzimidazol-6-yl)-N-(2-fluorophenyl)-2- methoxy-3-pyridinesulfonamide

[0134] The chemical structural formula of G1 is:

[0135]

[0136] (aS)-5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide.

[0137] The term "simeprevir" (C 38 H 47 N5O7S2), originally used for the treatment of HCV, later proved that simeprevir can also inhibit PI4KIIIa, and its inhibitory effect IC 50 200nM (Kwon J, Kim D, Park J, Park Y, Hwang Y, Wu H, Shin K, Kim I. Targeting Phosphatidylinositol 4-Kinase IIIa for Radiosensitization: A Potential Model of Drug Repositioning Using an Anti-Hepatitis C Viral Agent. Int J Radiation Oncol Biol Phys, 2016, Vol. 96(4) pp. 867-876).

[0138] This also relates to derivatives of phenarsazine, A1, G1 or analogs of simeprevir, as long as they have the function of inhibiting the phosphokinase activity of PI4KIIIa protein, and can also be used for the treatment of coronavirus diseases, especially the novel coronavirus, and the preparation method of such structural analogs has been disclosed. In some embodiments, the derivative of phenarsazine, A1, G1 or simeprevir is an analog similar in structure to phenarsazine, A1, G1 or simeprevir.

[0139] Coronaviruses

[0140] The term "coronavirus" (CoV) used herein refers to a class of single-stranded positive-sense RNA viruses with envelope, which can infect humans and various animals, has respiratory tract, gastrointestinal tract and nervous system tropism, can cause serious diseases in livestock and companion animals (such as pigs, cows, chickens, dogs, cats), and can cause humans to suffer from diseases from common cold to severe acute respiratory syndrome. According to the evolutionary characteristics of coronaviruses, the International Committee on Taxonomy of Viruses (ICTV) divides them into four groups: alpha, beta, gamma and 6, among which the hosts of alpha and beta groups are mainly mammals, and the hosts of gamma and 6 groups are mainly birds and poultry.

[0141] Coronaviruses that can infect humans include, but are not limited to, human coronavirus 229E (HCoV-229E), NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), OC43 (HCoV-OC43) that cause upper respiratory tract infection symptoms of the common cold, Severe acute respiratory syndrome coronavirus (SARS-CoV) and novel coronavirus (SARS-CoV-2) that can lead to severe respiratory illness, and Middle east respiratory syndrome coronavirus (MERS-CoV).

[0142] Coronaviruses that can infect animals include, but are not limited to, Transmissible gastroenteritis virus of swine (TGEV), porcine delta coronavirus (PDC), porcine hemagglutinating encephalomyelitis virus (PHEV), canine respiratory coronavirus (CrCoV), mouse hepatitis virus, feline coronavirus (FCoV).

[0143] Drug administration and medical uses

[0144] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. In certain embodiments, a pharmaceutically acceptable compound, material, composition, and / or dosage form is one which is approved by a regulatory agency of the Federal or a state government (such as the U.S. Food and Drug Administration, the China Food and Drug Administration, or the European Medicines Agency) or listed in the generally recognized pharmacopeia (e.g., U.S. Pharmacopeia, China Pharmacopeia, or European Pharmacopeia) for use in animals, and more particularly in humans.

[0145] The term "subject" as used herein can include both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals. A "subject" can also be a domestic animal (e.g., a cow, pig, sheep, chicken, rabbit, or horse), or a rodent (e.g., a rat or a mouse), or a primate (e.g., a great ape or monkey), or a pet (e.g., a dog or cat). A "subject" can be of either sex and of any age. A human "subject" can be Caucasian, African, Asian, Semitic, or of other race, or a hybrid of different races. A human "subject" can be geriatric, adult, adolescent, child, or infant.

[0146] In some embodiments, a subject described herein is a human or non-human primate.

[0147] The PI4KIIIa specific inhibitors disclosed herein can be administered by routes of administration known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection (including intravenous drip or infusion), intramuscular injection, or intradermal injection) or non-injection (e.g., oral administration, nasal administration, sublingual administration, rectal administration, or topical administration). In some embodiments, the PI4KIIIa specific inhibitors described herein are administered orally, subcutaneously, intramuscularly, or intravenously. In some embodiments, the PI4KIIIa specific inhibitors described herein are administered orally.

[0148] The term "therapeutically effective amount" as used herein refers to an amount of a drug that relieves or eliminates a disease or a symptom of a subject, or that preventsively inhibits or prevents the occurrence of a disease or a symptom. A therapeutically effective amount can be an amount of a drug that relieves a disease or a symptom of a subject to some extent; an amount of a drug that partially or completely restores one or more physiological or biochemical parameters associated with the cause of a disease or a symptom to normal; and / or an amount of a drug that reduces the likelihood of the occurrence of a disease or a symptom. In some embodiments, the term "therapeutically effective amount" as used herein refers to an amount of a drug that relieves or eliminates a coronavirus (e.g., a novel coronavirus) of a subject.

[0149] The therapeutically effective dose of the PI4KIIIa specific inhibitors provided herein depends on various factors known in the art, such as body weight, age, past medical history, current treatment, health status of the subject, and intensity of drug interaction, allergy, hypersensitivity, and side effects, as well as routes of administration and the extent of disease progression. A person skilled in the art (e.g., a physician or a veterinarian) can reduce or increase the dose according to these or other conditions or requirements.

[0150] In some embodiments, the treatment further comprises administering to a subject in need thereof a second agent.

[0151] In some embodiments, the second agent is an agent for treating a coronavirus disease, including but not limited to Lopinavir, Ritonavir, Remdesivir, Chloroquine, Simeprevir.

[0152] In some embodiments, the PI4KIIIa specific inhibitor is administered before, after, or simultaneously with the second agent.

[0153] The present application also relates to a method for preventing or treating a coronavirus disease, comprising administering to a subject in need thereof an effective amount of a PI4KIIIa specific inhibitor.

[0154] Drug screening

[0155] The present application also provides a method for screening a drug for preventing or treating a coronavirus disease, comprising contacting a candidate drug with a PI4KIIIa protein or nucleic acid or PI4KIIIa, and detecting whether the candidate drug can inhibit the formation or activity of PI4KIIIa.

[0156] Example 1. Solution preparation of phenarsazine and other compounds

[0157] In a weak light environment, an appropriate amount of PAO (phenarsazine, code PI01, synthesized by Shanghai Kaihui Pharmaceutical Co., Ltd.), YL-05, YL-07, YL-08, YL-09, YL-10, YL-11 (5 compounds were synthesized by BioChemPartner, the structural formula is as follows), Simeprevir (code PI0101), PI4KIIIa protein specific inhibitor PIK-93 (MedChemExpress), Remdesivir (Remdesivir) and Chloroquine phosphate (Chloroquine phosphate) and other compound powders were accurately weighed by an electronic balance (purchased from Mettler, Switzerland), dissolved with 5.0 mL of DMSO, and stored at a concentration of 20 mM or 40 mM of phenarsazine. The solution was filtered with a 0.22 μM filter to remove bacteria and stored at -20°C for standby, and attention should be paid to avoid light.

[0158]

[0159]

[0160] Example 2. CCK-8 cell proliferation-toxicity detection method for detecting the toxicity of different concentrations of phenarsazine to Vero cells

[0161] Vero cells (African green monkey kidney cells Vero, preserved by Zhejiang University National Key Laboratory of Infectious Disease Diagnosis and Treatment) were inoculated in a 96-well culture plate (purchased from Corning Company) at a concentration of 5000 cells / well, and cultured in a 37°C, 5% CO2 incubator (Thermo 3110, purchased from Thermo, USA) until the cell monolayer was formed. The culture solution was discarded, and the cells were washed twice with Hank's solution.

[0162] Drug solution dilution: In the first round of experiments, the concentrations of phenylarsenic oxide were diluted with MEM medium (purchased from Life Technologies Corporation) with a starting concentration of 200 nM and a continuous dilution of 7 concentrations to 1.56 nM. Each concentration of phenylarsenic oxide was set with 2 duplicate wells, and each well was added with 150 μL of drug solution. At the same time, a normal growth control of Vero cells was set, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours. In the second round of experiments, the starting concentrations of phenylarsenic oxide and PIK-93 were 800 nM, and a continuous dilution of 7 concentrations to 6.25 nM was performed. The starting concentration of Remdesivir was 300 μM, and a continuous dilution of 6 concentrations to 0.4 μM was performed. The starting concentration of Chloroquine was 300 μM, and a continuous dilution of 6 concentrations to 0.4 μM was performed. The starting concentration of PIK-93 was 800 nM, and a continuous dilution of 7 concentrations to 6.25 nM was performed. Each concentration of the drug was set with 2 duplicate wells, and each well was added with 150 μL of drug solution. At the same time, a normal growth control of Vero cells was set, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0163] CCK-8 reagent (cell proliferation-toxicity detection kit (CCK-8), purchased from Dojin Do Corporation of Japan) was added to each well at a volume of 10 or 15 μL, and after 3 hours, the OD value at 450 nm was determined by an enzyme-labeled instrument (Bio-Rad 680, purchased from BioRad Corporation of the United States) to calculate the dose toxicity effect of phenylarsenic oxide on cells and the maximum non-toxic concentration, and to draw a phenylarsenic oxide-cell toxicity response curve to provide a basis for the optimal test concentration selection of in vitro antiviral drug efficacy.

[0164] The first round of test results showed that phenylarsenic oxide at a concentration of 200 nM and above had a significant toxic effect on Vero cells, with cell death reaching 50%, i.e., the CC 50 was 200 nM. At a concentration of 100 nM or below, phenylarsenic oxide had no obvious toxic effect on cells, and the cell survival rate was greater than 90% (see Table 1A and Figure 1A ).

[0165] Table 1A: Toxic effect of different concentrations of phenylarsenic oxide on Vero cells

[0166]

[0167] The second round of test results showed that phenylarsenic oxide (PI01) drug at a concentration of 400 nM and above had a significant toxic effect on Vero cells, with cell death reaching 50% or more, and the CC 50 was 380 nM. At a concentration of 100 nM or below, the drug had no obvious toxic effect on cells, and the cell survival rate was greater than 90%, and the CC 10 was 100 nM.

[0168] 800nM and below concentration of PI4KIIIβ protein specific inhibitor PIK-93 did not have significant toxic effect on Vero cells.

[0169] Control drug Remdesivir, CC 50 was 208 μΜ, and the drug had no significant toxic effect on cells at a concentration of 50 μΜ and below, with cell viability greater than 90% above, CC 10 was 55 μΜ.

[0170] Control drug Chloroquine phosphate, CC 50 was about 250 μΜ, CC 10 was 120 μΜ.

[0171] The specific results are shown in

Table 1B, C, D and Figure 1B , C, D

[0172] Table 1B: Toxic effect of different concentrations of Oxidized phenylarsenic drug on Vero cells

[0173]

[0174] Table 1C: Toxic effect of different concentrations of Remdesivir drug on Vero cells

[0175]

[0176] Table 1D: Toxic effect of different concentrations of Chloroquine phosphate drug on Vero cells

[0177]

[0178]

[0179] Example 3. Determination of new coronavirus TCID by microcytopathic observation method 50

[0180] Vero cells were inoculated in 96-well culture plates at a concentration of 10000 cells / well, and cultured in a 37℃, 5% CO2 incubator to the most vigorous 75%-90% confluent cells in the logarithmic growth phase of the cells. The cell culture solution was discarded, and the cells were washed twice with Hank's solution (to remove residual bovine serum).

[0181] The new coronavirus was isolated from sputum samples of clinically infected persons and verified by whole genome sequencing, and was preserved by the National Key Laboratory of Infectious Disease Diagnosis and Treatment of Zhejiang University. The experiment was carried out in a biosafety level three laboratory (BSL-3: Laboratory Certification Number: CNAS BL002, National Key Laboratory of Infectious Disease Diagnosis and Treatment of Zhejiang University).

[0182] The new coronavirus strain virus growth fluid (500 ml MEM medium containing FBS 2%, penicillin 100 U / mL, chain enzyme 100 μg / mL, TPCK-trypsin 16 μg / mL) (all purchased from Life Technologies Company) was diluted 1000 times as the starting concentration, and then 10 times gradient dilution was performed for 8 series concentrations. Each virus dilution was inoculated into cells in 4 holes, 100 μl per hole, and normal cell controls were set up at the same time, 37°C, 5% CO2 incubator for 6 days.

[0183] The cell morphology and CPE changes were observed under an inverted microscope every day, and the CPE changes of 25% or less cell morphology were "+", 26%-50% cell morphology were "++", 51%-75% cell morphology were "+++", and 76%-100% cell morphology were "++++". The Reed-Muench method was used to calculate the TCID50 of the virus.

[0184] Results The TCID50 of the new coronavirus used in the experiment was 10 50 / 100 μL. -6.5

[0185] Example 4. Inhibition of new coronavirus (SARS-Cov-2) by phenarsazine in vitro

[0186] Cell culture: Vero cells were inoculated in 24-well plates at a concentration of 50000 / 1 mL / well, and incubated at 37°C in a 5% CO2 incubator until the cells reached the most vigorous logarithmic growth phase, 75%-90% of the cells were confluent, the cell culture medium was discarded, and the cells were washed twice with Hank's solution.

[0187] Drug solution preparation: The stored concentration of the drug was diluted with the medium to the following final concentrations: in the first round of experiments, the starting concentration of phenarsazine was 200 nM, and it was serially diluted by 2 to 7 concentrations to 1.56 nM; in the second round of experiments, the starting concentrations of phenarsazine and PIK-93 were 800 nM, and they were serially diluted by 2 to 7 concentrations to 6.25 nM; the starting concentration of Remdesivir was 100 μM, and it was serially diluted by 3 to 6 concentrations to 0.4 μM; the starting concentration of chloroquine phosphate was 100 μM, and it was serially diluted by 3 to 6 concentrations to 0.3 μM.

[0188] Virus solution preparation: The new coronavirus (SARS-CoV-2 strain BetaCov / Wuhan / IME-BJ01 / 2020 (GWHACBB01000000)) was diluted with the medium to a final concentration of 100 TCID 50 .​

[0189] Vero cells were infected with formulated virus (100 TCID 50 ) 250 μL / well. Virus-infected cell control and normal cell control were set up. After 3 hours of adsorption at 37 °C in a 5% CO2 incubator, the virus-containing culture solution was discarded and washed twice with Hank’s solution.

[0190] Drug treatment: Formulated culture solution containing different concentrations of drugs 1 mL / well was added to the above-mentioned pre-infected Vero cell culture plate, and two duplicate wells were set up for each concentration of phenarsazine. Incubation was continued at 37 °C in a 5% CO2 incubator for 48 hours.

[0191] The fluorescent quantitative PCR method was used to detect the nucleic acid of the novel coronavirus. 200 μL of culture supernatant was taken, and the viral nucleic acid was extracted using the magnetic bead method nucleic acid extraction kit (MVR01) and the automatic nucleic acid extraction instrument (EX3600, Shanghai Zhijiang Biotechnology Co., Ltd.). The final elution volume was 50 μl. 5 μL of nucleic acid extract was taken, and the one-step novel coronavirus nucleic acid detection kit (fluorescent PCR method, item number Z-RR-0479-02-50, national medical equipment standardization 20203400057, purchased from Shanghai Zhijiang Biotechnology Co., Ltd.) was used to detect the viral nucleic acid level. The Ct value was used to represent the level of the virus. The Ct value and the number of viral copies corresponding to the base 10 logarithm were as follows: y = -3.33x + 48.69, where y was the Ct value and x was the number of viral copies.

[0192] The results of the first round of experiments showed that phenarsazine had a significant antiviral effect on the Vero cell model at a concentration of 20-100 nM, with an EC 50 = 20 nM (see Table 2A and Figure 2A ). The FAM Ct value in Table 2 represents the viral nucleic acid level. The larger the Ct value, the fewer the viruses. Conversely, the smaller the Ct, the more viruses.

[0193] The results of the second round of experiments showed that the EC 50 = 19.2 nM for PI01 to inhibit the novel coronavirus. The EC 50 = 0.52 μM for Remdesivir to inhibit the novel coronavirus. The EC 50 = 50 μM for Chloroquine to inhibit the novel coronavirus. PIk-93 did not have a significant inhibitory effect on the novel coronavirus at a concentration of 800 nM or less. See Tables 2B, C, D and Figure 2B , C, D.

[0194] In addition, we also tested the inhibitory effect of PI0101 on the novel coronavirus. Cells were seeded into 96-well test plates at a density of 10,000 cells per well and incubated in a 5% CO2, 37°C incubator overnight. The next day, PI0101 was added at a 3-fold dilution (starting concentration of 10 mM, 8 concentration points, double duplicate wells), followed by virus at 100 TCID 50 Cells were added. Cell controls (cells, no compound treatment or virus infection), virus controls (cells infected with virus, no compound treatment), and media controls (media only) were set up and incubated in a 5% CO2, 37°C incubator for 76 hours. The results showed that the EC 50 = 3.85 mM, CC 50 was greater than 10 mM.

[0195] Table 2A: In vitro inhibition of the novel coronavirus by different concentrations of phenylarsenic oxide

[0196]

[0197] Table 2B: In vitro inhibition of the novel coronavirus by different concentrations of phenylarsenic oxide

[0198]

[0199] Table 2C: In vitro inhibition of the novel coronavirus by different concentrations of Remdesivir

[0200]

[0201] Table 2D: In vitro inhibition of the novel coronavirus by different concentrations of chloroquine phosphate

[0202]

[0203] Example 5. In vitro anti-human coronavirus (HCoV) 229E activity of phenylarsenic oxide detected by cytopathic effect (CPE) experiment

[0204] Drug solution preparation: The stock concentration of drugs were diluted to the following final concentrations with media separately. In the first round of experiments, the starting concentrations of Oxidized phenylarsenic, YL-05, YL-07, YL-08, YL-09, YL-10 and YL-11 compounds for toxicity to MRC5 cells and inhibition of HCoV 229E (Human coronavirus 229E type, purchased from ATCC) were 800 and 200 nM, respectively, and serially diluted by 2-fold to 7 concentrations of 6.25 and 1.56 nM, respectively; the starting concentrations of remdesivir for toxicity to MRC5 cells and inhibition of HCoV 229E were 100 μΜ and 1000 nM, respectively, and serially diluted by 3-fold to 7 concentrations of 0.41 μΜ and 0.46 nM, respectively.

[0205] In the second round of experiments, the starting concentrations of Oxidized phenylarsenic for cytotoxicity and viral inhibition were 800 and 400 nM, respectively, and serially diluted by 2-fold to 7 concentrations of 6.25 and 3.13 nM, respectively; the starting concentrations of YL-07, YL-08, YL-09, YL-10 and YL-11 compounds for toxicity to MRC5 cells and inhibition of HCoV 229E were all 40 μΜ, and serially diluted by 3-fold to 7 concentrations of 0.02 μΜ; the starting concentrations of remdesivir for cytotoxicity and viral inhibition were 100 μΜ and 1000 nM, respectively, and serially diluted by 3-fold to 7 concentrations of 0.41 μΜ and 0.46 nM, respectively.

[0206] MRC5 cells and HCoV 229E strain were purchased from ATCC. Cells were cultured with EMEM (Sigma) medium added with 10% fetal bovine serum (Hyclone), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco). EMEM (Sigma) medium added with 5% fetal bovine serum (Hyclone), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco) was used as experimental medium.

[0207] MRC5 cells were seeded into 96-well test plates at a density of 20,000 cells per well and cultured in a 5% CO2, 37°C incubator overnight. The next day, Oxidized phenylarsenic was added with 8 concentration points (double duplicate wells) followed by virus at 200 TCID 50Cells were added. Cell controls (cells, no compound treatment or viral infection), virus controls (cells infected with virus, no compound treatment), and culture medium controls (culture medium only) were set up. The final concentration of DMSO in the culture medium was 0.5%. Cells were cultured in an incubator for 3 days. Cell viability was assessed using the CellTiter Glo (Promega) cell viability assay kit. Cytotoxicity assays were performed under the same conditions as antiviral assays, but without viral infection. The antiviral activity and cytotoxicity of arsenic oxide were expressed as the inhibition rate (%) of the compound at different concentrations against virus-induced cytopathic effects and the viability (%) of MRC5 cells, respectively. The calculation formulas are as follows:

[0208] Inhibition rate (%) = (Test well reading - Average value of virus control) / (Average value of cell control - Average value of virus control) x 100

[0209] Cell viability (%) = (Test well reading - Average value of culture medium control) / (Average value of cell control - Average value of culture medium control) x 100

[0210] The inhibition rate and cell viability of arsenic oxide were analyzed using nonlinear fitting analysis with GraphPad Prism (version 5) to calculate the half-maximal effective concentration (EC50) of arsenic oxide. 50 ).

[0211] The first round of results showed that the EC50 of phenylarsine oxide... 50 The value is 55.35 nM, CC 50 The value was 256.8 nM; Remdesivir EC 50 The value is 26.42 nM, CC 50 Values ​​greater than 100 μM; EC values ​​of compounds YL-05, YL-07, YL-08, YL-09, YL-10, and YL-11 50 All are greater than 200 nm, CC 50 All are greater than 800 nM; please see the detailed results. Figure 3A And Table 3. The second round of results shows that the EC of oxidized phenylarsine... 50 The value is 37.03 nM, CC 50 The value was 68.92 nM; Remdesivir EC 50 The value is 13.11 nM, CC 50 The value was 30.66 μM; EC values ​​for compounds YL-07, YL-08, and YL-09 were... 50 All are greater than 40μM, CC 50 The EC values ​​for compounds YL-10 and YL-11 were 6.24 μM, 7.64 μM, and 13.88 μM, respectively. 50 The concentrations were 3.95 μM and 2.1 μM, respectively, CC 5014 μM and 6.37 μM, respectively; see Table 3 for specific results Figure 3B and Table 3. The EC50for PI0101 (simeprevir) was 3.41 μM, and the CC50was 8.46 μM. 50 and Table 3. The EC50for PI0101 (simeprevir) was 3.41 μM, and the CC50was 8.46 μM. 50 and Table 3. The EC50for PI0101 (simeprevir) was 3.41 μM, and the CC50was 8.46 μM.

[0212] Table 3. Compound test results

[0213]

[0214] Note: ND, not determined

[0215] It should be understood that while particular embodiments of the present application have been described herein in detail, various modifications can be made without departing from the spirit and scope of the present application. Therefore, the present application should not be considered to be limited to the particular embodiments described herein, but rather should be understood to cover all modifications that fall within the scope of the present application. The present application is limited only by the claims appended hereto. All documents cited herein are hereby fully incorporated by reference.

Claims

1. Use of a PI4KIIIa specific inhibitor in the manufacture of a medicament for treating a coronavirus disease, wherein the PI4KIIIa specific inhibitor is phenarsazine or simeprevir, and the coronavirus is a novel coronavirus or human coronavirus 229E.

2. The use of claim 1, wherein the subject of treatment is a human or a mammal.

3. The use of claim 1 or 2, further comprising administering to a subject in need thereof a second agent, wherein the second agent is an agent for treating a coronavirus disease.

4. The use of claim 3, wherein the PI4KIIIa specific inhibitor is administered prior to, after, or concurrently with the second agent.

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