Protozoan transcription factor inhibitors

Abstract

The present disclosure provides an antiprotozoal agent targeting a transcription factor.　The present disclosure provides a pyrrole-imidazole polyamide (PIPA) specifically binding to a binding region of a protozoa transcription factor.

Description

[Technical Field]

[0001] The present disclosure relates to pyrrole-imidazole polyamides (PIPAs) that specifically bind to the binding domains of protozoan transcription factors, a method for producing the PIPAs, and protozoan transcription factor inhibitors containing the PIPAs. More specifically, the present disclosure relates to a technology for treating or preventing diseases caused by protozoan parasites by inhibiting morphological changes in the protozoan parasites using PIPAs that can function as competitive pseudo-transcription factors that specifically bind to the binding domains of the protozoan transcription factors. [Background technology]

[0002] Protozoan infections such as malaria are infectious diseases that have yet to be eradicated. For example, in the case of malaria, it is reported that 500,000 people die each year worldwide. Furthermore, despite the ongoing development of new drugs, drug-resistant strains emerge after a certain period of time, making a complete cure difficult. Summary of the Invention [Means for solving the problem]

[0003] Although protozoa are eukaryotic organisms, they possess highly distinctive transcriptional regulatory mechanisms compared to mammalian cells. To date, no drugs targeting protozoan transcription factors have been developed. This disclosure provides an antiprotozoal drug targeting transcription factors by using pyrrole-imidazole polyamides (PIPAs) that can function as pseudo-transcription factors.

[0004] Thus, the present disclosure provides: (Item 1) Pyrrole-imidazole polyamide (PIPA) specifically binds to the binding domain of a protozoan transcription factor. (Item 2) A pyrrole-imidazole polyamide (PIPA) according to the above item, which specifically binds to the binding region of a protozoan transcription factor, wherein the protozoan transcription factor is a transcription factor specific to protozoa. (Item 3) The PIPA according to any one of the preceding items, which functions as a pseudo-transcription factor. (Item 4) The PIPA according to any one of the preceding items, which has a binding affinity to the binding domain as expressed as a dissociation constant (Kd value) of about 500 nM or less. (Item 5) The PIPA described in any one of the above items, wherein the PIPA has a hairpin structure or a cyclic structure, or two linear PIPAs are used in combination. (Item 6) The PIPA described in any one of the above items, which inhibits the morphological transformation of the protozoan at least into gametocytes. (Item 7) The PIPA of any one of the preceding items, wherein the transcription factor comprises an AP2 family transcription factor. (Item 8) A PIPA described in any one of the above items, wherein the binding region comprises 5'-TGCATG-3' (SEQ ID NO: 1) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCATG-3' (SEQ ID NO: 1) is deleted or mutated, and a sequence in which one base is added to any position in 5'-TGCATG-3' (SEQ ID NO: 1). (Item 9) The PIPA of any one of the preceding items, wherein the binding region comprises NGCATG (SEQ ID NO: 2), TNCATG (SEQ ID NO: 3), TGNATG (SEQ ID NO: 4), TGCNTG (SEQ ID NO: 5), TGCANG (SEQ ID NO: 6), and TGCATN (SEQ ID NO: 7), wherein N is A, T, G, or C. (Item 10) A PIPA described in any one of the above items, wherein the binding region comprises 5'-TGCACT-3' (sequence number 8) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCACT-3' (sequence number 8) is deleted or mutated, and a sequence in which one base is added to any position in 5'-TGCACT-3' (sequence number 8). (Item 11) The PIPA of any one of the preceding items, wherein the binding region comprises NGCACT (SEQ ID NO: 9), TNCACT (SEQ ID NO: 10), TGNACT (SEQ ID NO: 11), TGCNCT (SEQ ID NO: 12), TGCANT (SEQ ID NO: 13), and TGCACN (SEQ ID NO: 14), and N is A, T, G, or C. (Item 12) The following structure: [ka] or [ka] where: L is a C2-6 alkyl linker; R1 and R2 are optionally substituted alkyl, and R1 and R2 together may form a C2-6 alkyl linker; The PIPA of any one of the preceding items, wherein X is a bond or an aliphatic amino acid residue. (Item 13) The PIPA according to any one of the preceding items, wherein the aliphatic amino acid residue comprises a molecule having an amino group and a carboxy group. (Item 14) The PIPA of any one of the preceding items, wherein the aliphatic amino acid residues include glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid. (Item 15) The following structure: [ka] The PIPA according to any one of the preceding items, (Item 16) The following structure: [ka] The PIPA according to any one of the preceding items, (Item 17) The PIPA of any one of the preceding items, wherein the protozoa include malaria, leishmania, toxoplasma, cryptosporidium, and coccidia. (Item A1) Pyrrole-imidazole polyamides (PIPA) that specifically bind to the binding domains of protozoan transcription factors to inhibit their function. (Item A2) The PIPA according to any one of the preceding items, wherein the protozoan transcription factor is a transcription factor specific to the protozoan. (Item A3) The PIPA according to any one of the preceding items, which functions as a pseudo-transcription factor. (Item A4) The PIPA according to any one of the preceding items, which has a binding affinity to the binding domain as expressed as a dissociation constant (Kd value) of about 500 nM or less. (Item A5) The PIPA described in any one of the above items, wherein the PIPA has a hairpin structure or a cyclic structure, or two linear PIPAs are used in combination. (Item A6) The PIPA described in any one of the above items, which inhibits the morphological transformation of the protozoan at least into gametocytes. (Item A7) The PIPA of any one of the preceding items, wherein the transcription factor comprises an AP2 family transcription factor. (Item A8) A PIPA described in any one of the above items, wherein the binding region comprises 5'-TGCATG-3' (SEQ ID NO: 1) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCATG-3' (SEQ ID NO: 1) is deleted or mutated, and a sequence in which one base is added to any position in 5'-TGCATG-3' (SEQ ID NO: 1). (Item A9) The PIPA of any one of the preceding items, wherein the binding region comprises NGCATG (SEQ ID NO: 2), TNCATG (SEQ ID NO: 3), TGNATG (SEQ ID NO: 4), TGCNTG (SEQ ID NO: 5), TGCANG (SEQ ID NO: 6), and TGCATN (SEQ ID NO: 7), wherein N is A, T, G, or C. (Item A10) A PIPA described in any one of the above items, wherein the binding region comprises 5'-TGCACT-3' (sequence number 8) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCACT-3' (sequence number 8) is deleted or mutated, and a sequence in which one base is added to any position in 5'-TGCACT-3' (sequence number 8). (Item A11) The PIPA of any one of the preceding items, wherein the binding region comprises NGCACT (SEQ ID NO: 9), TNCACT (SEQ ID NO: 10), TGNACT (SEQ ID NO: 11), TGCNCT (SEQ ID NO: 12), TGCANT (SEQ ID NO: 13), and TGCACN (SEQ ID NO: 14), and N is A, T, G, or C. (Item A12) The following structure: [ka] or [ka] where: L is a C2-6 alkyl linker; R1 and R2 are optionally substituted alkyl, and R1 and R2 together may form a C2-6 alkyl linker; The PIPA of any one of the preceding items, wherein X is a bond or an aliphatic amino acid residue. (Item A13) The PIPA of any one of the preceding items, wherein the aliphatic amino acid residue comprises a molecule having an amino group and a carboxy group. (Item A14) The PIPA of any one of the preceding items, wherein the aliphatic amino acid residues include glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid. (Item A15) The following structure: [ka] The PIPA according to any one of the preceding items, (Item A16) The following structure: [ka] The PIPA according to any one of the preceding items, (Item A17) The PIPA of any one of the preceding items, wherein the protozoa include malaria, leishmania, toxoplasma, cryptosporidium, and coccidia. (Item A1A) Pyrrole-imidazole polyamides (PIPA) that specifically bind to the binding domains of protozoan transcription factors for use as pseudo-transcription factors. (Item A1B) A composition comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor for inhibiting the function of the protozoan transcription factor. (Item A1C) A composition comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor for use as a pseudo-transcription factor. (Item A1D) A method for inhibiting the function of a protozoan transcription factor in a subject, comprising contacting the protozoan transcription factor in the subject with an effective amount of a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of the protozoan transcription factor. (Item A1E) A method for using pyrrole-imidazole polyamide (PIPA) as a pseudo-transcription factor in a subject, comprising the step of applying to the subject an effective amount of PIPA that specifically binds to the binding domain of a protozoan transcription factor. (Item B1) A protozoan transcription factor inhibitor containing pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor. (Item B2) The protozoan transcription factor inhibitor according to any one of the above items, wherein the protozoan transcription factor is a transcription factor specific to the protozoan. (Item B3) The protozoan transcription factor inhibitor according to any one of the above items, which functions as a pseudo-transcription factor (Item B4). The protozoan transcription factor inhibitor according to any one of the preceding items, which has a binding affinity to the binding domain as expressed as a dissociation constant (Kd value) of about 500 nM or less. (Item B5) The protozoan transcription factor inhibitor according to any one of the above items, wherein the PIPA has a hairpin structure or a cyclic structure, or two linear PIPAs are used in combination (Item B6). The protozoan transcription factor inhibitor according to any one of the above items, which inhibits at least the morphological transformation of the protozoan into gametocytes. (Item B7) The protozoan transcription factor inhibitor according to any one of the preceding items, wherein the transcription factor comprises an AP2 family transcription factor. (Item B8) A protozoan transcription factor inhibitor described in any one of the above items, wherein the binding region comprises 5'-TGCATG-3' (SEQ ID NO: 1) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCATG-3' (SEQ ID NO: 1) is deleted or mutated, and a sequence in which one base is added to any position in 5'-TGCATG-3' (SEQ ID NO: 1). (Item B9) A protozoan transcription factor inhibitor described in any one of the above items, wherein the binding region comprises NGCATG (sequence number 2), TNCATG (sequence number 3), TGNATG (sequence number 4), TGCNTG (sequence number 5), TGCANG (sequence number 6), and TGCATN (sequence number 7), and N is A, T, G, or C. (Item B10) A protozoan transcription factor inhibitor described in any one of the above items, wherein the binding region comprises 5'-TGCACT-3' (sequence number 8) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCACT-3' (sequence number 8) is deleted or mutated, and a sequence in which one base is added to any position in 5'-TGCACT-3' (sequence number 8). (Item B11) A protozoan transcription factor inhibitor described in any one of the above items, wherein the binding region comprises NGCACT (SEQ ID NO: 9), TNCACT (SEQ ID NO: 10), TGNACT (SEQ ID NO: 11), TGCNCT (SEQ ID NO: 12), TGCANT (SEQ ID NO: 13), and TGCACN (SEQ ID NO: 14), and N is A, T, G, or C. (Item B12) The following structure: [ka] or [ka] where: L is a C2-6 alkyl linker; R1 and R2 are optionally substituted alkyl, and R1 and R2 together may form a C2-6 alkyl linker; The protozoan transcription factor inhibitor according to any one of the preceding items, wherein X is a bond or an aliphatic amino acid residue. (Item B13) The protozoan transcription factor inhibitor according to any one of the preceding items, wherein the aliphatic amino acid residue comprises a molecule having an amino group and a carboxy group. (Item B14) The protozoan transcription factor inhibitor according to any one of the preceding items, wherein the aliphatic amino acid residues include glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid. (Item B15) The following structure: [ka] The protozoan transcription factor inhibitor according to any one of the above items, (Item B16) The following structure: [ka] The protozoan transcription factor inhibitor according to any one of the above items, (Item B17) The protozoan transcription factor inhibitor according to any one of the preceding items, wherein the protozoan includes malaria, leishmania, toxoplasma, cryptosporidium, and coccidia. (Item C1) A method for producing a protozoan transcription factor inhibitor comprising pyrrole-imidazole polyamide (PIPA), comprising: providing a binding domain for a protozoan transcription factor; designing a PIPA to specifically bind to the binding domain; A method comprising: (Item C2) The designing step includes linking pyrroles and / or imidazoles selected to correspond to the nucleotide sequence of the binding region, and, if necessary, substituting β-alanine for one or more pyrroles or imidazoles in the linked pyrrole and / or imidazole molecules. (Item D1) A therapeutic or preventive agent for a disease caused by protozoa, comprising a protozoan transcription factor inhibitor, wherein the protozoan transcription factor inhibitor comprises a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding region of the protozoan transcription factor. (Item D2) The therapeutic or preventive agent according to any one of the preceding items, wherein the protozoan transcription factor is a transcription factor specific to protozoa. (Item D3) The therapeutic or prophylactic agent according to any one of the preceding items, which functions as a pseudo-transcription factor. (Item D4) The therapeutic or prophylactic agent according to any one of the preceding items, which has a binding affinity for the binding domain as expressed as a dissociation constant (Kd value) of about 500 nM or less. (Item D5) The therapeutic or prophylactic agent according to any one of the preceding items, wherein the PIPA has a hairpin structure or a cyclic structure, or two linear PIPAs are used in combination. (Item D6) The therapeutic or preventive agent according to any one of the preceding items, which inhibits at least the morphological transformation of the protozoan into gametocytes. (Item D7) The therapeutic or prophylactic agent according to any one of the preceding items, wherein the transcription factor comprises an AP2 family transcription factor. (Item D8) The therapeutic or preventive agent according to any one of the preceding items, wherein the binding region comprises 5'-TGCATG-3' (SEQ ID NO: 1) or a modified sequence thereof, and the modified sequence comprises a sequence in which any one base in 5'-TGCATG-3' (SEQ ID NO: 1) has been deleted or mutated, and a sequence in which one base has been added to any position in 5'-TGCATG-3' (SEQ ID NO: 1). (Item D9) The therapeutic or prophylactic agent according to any one of the preceding items, wherein the binding region comprises NGCATG (SEQ ID NO: 2), TNCATG (SEQ ID NO: 3), TGNATG (SEQ ID NO: 4), TGCNTG (SEQ ID NO: 5), TGCANG (SEQ ID NO: 6), and TGCATN (SEQ ID NO: 7), wherein N is A, T, G, or C. (Item D10) The therapeutic or preventive agent according to any one of the preceding items, wherein the binding region comprises 5'-TGCACT-3' (SEQ ID NO: 8) or a modified sequence thereof, and the modified sequence comprises a sequence in which any one base in 5'-TGCACT-3' (SEQ ID NO: 8) has been deleted or mutated, and a sequence in which one base has been added to any position in 5'-TGCACT-3' (SEQ ID NO: 8). (Item D11) The therapeutic or prophylactic agent according to any one of the preceding items, wherein the binding region comprises NGCACT (SEQ ID NO: 9), TNCACT (SEQ ID NO: 10), TGNACT (SEQ ID NO: 11), TGCNCT (SEQ ID NO: 12), TGCANT (SEQ ID NO: 13), and TGCACN (SEQ ID NO: 14), and N is A, T, G, or C. (Item D12) The following structure: [ka] or [ka] where: L is a C2-6 alkyl linker; R1 and R2 are optionally substituted alkyl, and R1 and R2 together may form a C2-6 alkyl linker; The therapeutic or prophylactic agent according to any one of the preceding items, wherein X is a bond or an aliphatic amino acid residue. (Item D13) The therapeutic or prophylactic agent according to any one of the preceding items, wherein the aliphatic amino acid residue comprises a molecule having an amino group and a carboxy group. (Item D14) The therapeutic or prophylactic agent according to any one of the preceding items, wherein the aliphatic amino acid residues include glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid. (Item D15) The following structure: [ka] The therapeutic or prophylactic agent according to any one of the above items, (Item D16) The following structure: [ka] The therapeutic or prophylactic agent according to any one of the above items, (Item D17) The therapeutic or preventive agent according to any one of the preceding items, wherein the protozoa include malaria, leishmania, toxoplasma, cryptosporidium, and coccidium. (Item D1A) A pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor for treating or preventing diseases caused by protozoa. (Item E1) A method for treating or preventing a disease caused by a protozoan in a subject, comprising administering to the subject an effective amount of a protozoan transcription factor inhibitor, wherein the protozoan transcription factor inhibitor comprises a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of the protozoan transcription factor. (Item X1) A conjugate comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding region of a protozoan transcription factor, and a protozoan-specific factor different from the PIPA. (Item X2) The conjugate according to any one of the preceding items, wherein the protozoan-specific factor comprises a factor that binds to a surface protein of malaria-infected red blood cells. (Item X3) The conjugate according to any one of the preceding items, wherein the protozoan-specific factor has an inhibitory effect on the growth of malaria, leishmania, toxoplasma, cryptosporidium, and / or coccidia. (Item X4) The conjugate according to any one of the preceding items, wherein the PIPA and the protozoan-specific factor are linked via a linker. (Item X5) The conjugate according to any one of the preceding items, wherein the linker is a C1-6 alkyl linker. (Item X6) The conjugate according to any one of the preceding items, wherein the PIPA and the protozoan-specific factor are directly linked. (Item X7) The conjugate according to any one of the preceding items, wherein the protozoan-specific factor comprises a pyridazinone derivative. (Item X8) The conjugate according to any one of the preceding items, wherein the pyridazinone derivative is MBX-4055 represented by the following formula: JPEG0007756380000017.jpg51127 (Item X9) The conjugate according to any one of the preceding items, wherein the pyridazinone derivative is an MBX-4055 derivative represented by the following formula: JPEG0007756380000018.jpg48127 (Item X10) The conjugate according to any one of the preceding items, wherein the pyridazinone derivative is an MBX-4055 derivative represented by the following formula: JPEG0007756380000019.jpg44127 (Item X11) The conjugate according to any one of the preceding items, wherein the pyridazinone derivative is an MBX-4055 derivative represented by the following formula: JPEG0007756380000020.jpg41127 (Item X12) The conjugate according to any one of the preceding items, wherein the protozoan transcription factor is a transcription factor specific to a protozoan. (Item X13) The conjugate according to any one of the preceding items, which functions as a pseudo-transcription factor. (Item X14) The conjugate according to any one of the preceding items, which has a binding affinity to the binding region as expressed as a dissociation constant (Kd value) of about 500 nM or less. (Item X15) The protozoan transcription factor inhibitor according to any one of the preceding items, wherein the PIPA has a hairpin structure or a cyclic structure, or two linear PIPAs are used in combination. (Item X16) The conjugate according to any one of the preceding items, which inhibits the morphological transformation of the protozoan into at least gametocytes. (Item X17) The conjugate according to any one of the preceding items, wherein the transcription factor comprises an AP2 family transcription factor. (Item X18) The conjugate according to any one of the preceding items, wherein the binding region comprises 5'-TGCATG-3' (SEQ ID NO: 1) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCATG-3' (SEQ ID NO: 1) has been deleted or mutated, and a sequence in which one base has been added to any position in 5'-TGCATG-3' (SEQ ID NO: 1). (Item X19) The conjugate of any one of the preceding items, wherein the binding region comprises NGCATG (SEQ ID NO: 2), TNCATG (SEQ ID NO: 3), TGNATG (SEQ ID NO: 4), TGCNTG (SEQ ID NO: 5), TGCANG (SEQ ID NO: 6), and TGCATN (SEQ ID NO: 7), wherein N is A, T, G, or C. (Item X20) The conjugate according to any one of the preceding items, wherein the binding region comprises 5'-TGCACT-3' (SEQ ID NO: 8) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-TGCACT-3' (SEQ ID NO: 8) has been deleted or mutated, and a sequence in which one base has been added to any position in 5'-TGCACT-3' (SEQ ID NO: 8). (Item X21) The conjugate of any one of the preceding items, wherein the binding region comprises NGCACT (SEQ ID NO: 9), TNCACT (SEQ ID NO: 10), TGNACT (SEQ ID NO: 11), TGCNCT (SEQ ID NO: 12), TGCANT (SEQ ID NO: 13), and TGCACN (SEQ ID NO: 14), and N is A, T, G, or C. (Item X22) PIPA has the following structure: JPEG0007756380000021.jpg102153 or JPEG0007756380000022.jpg108153, where: L is a C2-6 alkyl linker; R1 and R2 are optionally substituted alkyl, and R1 and R2 together may form a C2-6 alkyl linker; The conjugate of any one of the preceding items, wherein X is a bond or an aliphatic amino acid residue. (Item X23) The conjugate according to any one of the preceding items, wherein the aliphatic amino acid residue comprises a molecule having an amino group and a carboxy group. (Item X24) The conjugate of any one of the preceding items, wherein the aliphatic amino acid residues include glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid. (Item X25) PIPA has the following structure: JPEG0007756380000023.jpg91153 The conjugate according to any one of the preceding items, (Item X26) PIPA has the following structure: The conjugate according to any one of the preceding items, wherein the conjugate is JPEG0007756380000024.jpg64153. (Item X27) The conjugate of any one of the preceding items, wherein the protozoan includes malaria, leishmania, toxoplasma, cryptosporidium, and coccidia.

[0005] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] Note that features and significant actions and effects of the present disclosure other than those described above will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings. [Effects of the Invention]

[0007] The present disclosure provides PIPA that specifically binds to the binding domain of a protozoan transcription factor, which can provide an agent for treating or preventing protozoan-induced diseases that are still difficult to cure, and an innovative and powerful drug for eradicating protozoan infections. [Brief explanation of the drawings]

[0008] [Figure 1a] 1a shows the sequence, structural formula, and molecular weight of PIPA according to one embodiment of the present disclosure, in which AP2-PIPA1 (FIG. 1a, left) and AP2-PIPA2 (FIG. 1a, right) used in Example 1 are shown. [Figure 1b] FIG. 1b is a schematic diagram showing DNA fragments used in analyzing the binding strength of AP2-PIPA1 to a target sequence in one embodiment of the present disclosure. [Figure 1c] FIG. 1c is a graph showing the analysis results of the binding affinity (Kd value) of AP2-PIPA1 to a target sequence in one embodiment of the present disclosure. [Figure 2a] FIG. 2a is a graph showing the analysis results of the cytotoxicity of AP2-PIPA1 (DNA release in the culture supernatant) in one embodiment of the present disclosure. [Figure 2b]FIG. 2b is a graph showing the analysis results of the cytotoxicity of AP2-PIPA1 (LDH activity in the culture supernatant) in one embodiment of the present disclosure. [Figure 3a] FIG. 3 a is a micrograph showing the results of cell analysis of the malaria inhibitory effects of AP2-PIPA1 and AP2-PIPA2 in one embodiment of the present disclosure. [Figure 3b] FIG. 3b shows an illustration of cell analysis (FACS analysis) of the malaria inhibitory effects of AP2-PIPA1 and AP2-PIPA2 in one embodiment of the present disclosure. [Figure 3c] FIG. 3c is a graph showing the results of cell analysis (FACS analysis) of the malaria inhibitory effects of AP2-PIPA1 and AP2-PIPA2 in one embodiment of the present disclosure. [Figure 4] 4 is a graph showing the results of a cellular analysis of the inhibitory effects of AP2-PIPA1 and AP2-PIPA2 on drug-resistant malaria strains in one embodiment of the present disclosure. Malaria was cultured in red blood cells in the presence of various concentrations of PIPA, and parasitemia was assessed 72 hours later. [Figure 5a] Figure 5a shows the analysis results of the toxicity of AP2-PIPA1 in mice in one embodiment of the present disclosure. The toxicity of AP2-PIPA1 following a single intraperitoneal administration was examined using mice. The doses were 0 (PBS), 5, 10, and 20 mg / kg, and the mice were observed for 7 days after administration. Toxicity assessment included observation of viability and general condition, weight measurement, hematology, blood chemistry, and necropsy. No deaths were observed, and no abnormal changes were observed in general condition observation, weight change, hematology, blood chemistry, or necropsy. Significant increases in AST activity were observed in the 5 and 20 mg / kg groups. Although no significant differences were observed, there was an increasing trend in the 10 mg / kg group. However, individual values ​​in these groups were within the background range (34-58 IU / L), suggesting this change was due to physiological fluctuations. Based on these results, the minimum lethal dose of AP2-PIPA1 in this study was estimated to be greater than 20 mg / kg. [Figure 5b]Figure 5b shows the results of an analysis of the toxicity of AP2-PIPA1 in mice in one embodiment of the present disclosure. Mice were orally or intraperitoneally administered various doses of AP2-PIPA1 once daily for 7 days. Blood was collected on day 8, and AST and ALT were measured. Significance tests were performed on the 3 mg / kg and 10 mg / kg groups compared to the control group for each administration route. Bartlett's equal variance test showed p>0.01, indicating equal variance. Dunnett's two-tailed test showed p>0.05, indicating no significant difference. [Figure 6] FIG. 6 shows an analysis of the malaria-inhibitory effect of AP2-PIPA1 in mice in one embodiment of the present disclosure. [Figure 7a] 7a shows the sequence, structural formula, and molecular weight of PIPA that targets TGCATG in one embodiment of the present disclosure (the positions of β-alanine were modified). The positions of two β-alanine residues in AP2-PIPA1 were modified. [Figure 7b] Figure 7b shows the sequence, structural formula, and molecular weight of PIPA targeting TGCATG in one embodiment of the present disclosure (one base pair of pyrrole was added). Because AP2-PIPA1 uses γAbu, which has affinity for A-T pairs in its hairpin structure, a Py-Py pair was added to recognize this A-T pair. When the recognition sequence is increased and the 5' end is a GC / CG pair, γAbu is not involved in binding. [Figure 7c] Figure 7c shows the sequence, structural formula, and molecular weight of PIPA targeting TGCATG in one embodiment of the present disclosure (the hairpin structure was converted from γAbu to D-Dab). Because AP2-PIPA1 uses γAbu, which has affinity for A-T pairs, in its hairpin structure, this hairpin structure was converted to D-Dab. D-Dab also has affinity for A-T pairs. [Figure 8] FIG. 8 is a graph showing the therapeutic effect of malaria by PIPA of the present disclosure in humanized mice in one embodiment of the present disclosure. [Figure 9] FIG. 9 is a diagram showing a schematic diagram of a conjugate of the present disclosure and the structures of MBX-4055 and its derivatives therefor in one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0010] As used herein, the term "pseudo-transcription factor" is broadly interpreted to refer to a substance that has the property of binding to a conserved sequence to which a particular transcription factor specifically binds, and inhibits transcription and / or activation of the transcription factor via that binding sequence.

[0011] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0012] As used herein, "about" means ±10% of the preceding numerical value.

[0013] As used herein, "pyrrole-imidazole polyamide (PIPA)" refers to a low-molecular-weight organic compound primarily composed of pyrrole- and imidazole-containing amino acid residues. PIPA binds to double-stranded DNA more strongly and sequence-specifically than transcription factors, potently suppressing the transcriptional activity of target genes. The surface of the DNA double helix contains two grooves: a deep and a shallow groove. PIPA enters the shallow groove (minor groove) and reversibly binds to each base of the DNA via hydrogen bonds. Because PIPA is a condensation product of amino acids, it can be considered a polypeptide. However, because it is a completely artificial compound, it is resistant to degradation by various proteolytic enzymes in vivo and is stable in vivo. Furthermore, its ability to easily cross biological membranes eliminates the need for a drug delivery system (DDS).

[0014] As used herein, the term "protozoan" is broadly interpreted to refer to organisms that infect humans and other animals and cause harm to the infected subject. For example, protozoans include, but are not limited to, pathogens that cause malaria, leishmania, toxoplasma, cryptosporidium, coccidiosis, babesia, theileria, cystoisospora, and other protozoan infections.

[0015] As used herein, the term "protozoan transcription factor" refers to a transcription factor that functions within the body of a protozoan. In a narrow sense, it refers to a transcription factor that is specific to a protozoan and functions only in the protozoan, and in a broad sense, it also includes the general transcription factors described below.

[0016] As used herein, the term "general transcription factor" refers to a transcription factor common to all eukaryotes, including protozoa. It is a factor required for RNA polymerase to correctly recognize a promoter and initiate transcription. There are six types of general transcription factors required for transcription by RNA polymerase II, for example (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH). Although there are differences depending on the species, all organisms possess proteins similar to these. The recognition sequence is generally a sequence containing TATA, known as the TATA box, but it is also possible for specific sequences other than the TATA box to be recognized.

[0017] As used herein, the term "protozoan transcription factor binding region" refers to a genomic DNA sequence or region thereof to which a protozoan transcription factor binds.

[0018] As used herein, "specific binding" refers to the binding of molecules or substances to each other under conditions in which they do not substantially bind significantly to other molecules or substances. "Specific binding" refers to a binding reaction in which the PIPA of the present disclosure has a binding affinity of about 500 nM or less as a dissociation constant (Kd value). "Specifically" preferably means that the level of binding to molecules other than the target molecule results in a binding affinity of about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, and more preferably about 5% or less of the affinity to the target molecule. Any method known in the art can be used to measure the dissociation constant, and for example, the method exemplified in the Examples can be used. As used herein, a PIPA that specifically binds to a certain binding region can have a corresponding PIPA structure identified as long as the nucleic acid sequence of the target binding region is fixed. This identification can be achieved, for example, by referring to Journal of the American Chemical Society, 2012, vol. 134, pp. 17814-17822, etc. A person skilled in the art can design such a circuit appropriately, and the description thereof is given in other parts of this specification.

[0019] As used herein, "treatment" refers broadly to either preventive and / or therapeutic treatment, and in a narrower sense to alleviating, attenuating, or improving at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, for example, suppressing the onset of a disease or condition, alleviating a disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or arresting the symptoms of a disease or condition, for the purpose of improving (curing) the pathological condition. As used herein, "treatment" refers to alleviating, attenuating, or improving at least one symptom of a disease or condition, for the purpose of improving (curing) the pathological condition.

[0020] As used herein, "prevention" refers to preventing the development of clinical symptoms of a disease state in a subject who may be exposed to or susceptible to the disease state but who has not yet experienced or exhibited symptoms of the disease state.

[0021] As used herein, the term "gene" refers to a factor that determines a genetic trait. It may refer to a nucleic acid itself, a "polynucleotide," an "oligonucleotide," an "RNA," or a "DNA," and may also refer to a protein, polypeptide, oligopeptide, or peptide encoded by a nucleic acid. Those skilled in the art can appropriately understand this term depending on the context. Genes encoding such proteins may be endogenous or exogenous to the target organism. Furthermore, known genes may be used as appropriate. Genes may be used regardless of their origin. That is, genes may be derived from organisms of other species or genus than the target organism, or from organisms such as animals, plants, fungi (molds), and bacteria. Those skilled in the art can appropriately obtain information on such genes by accessing websites such as NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov). These genes may also be genes encoding proteins that have a certain relationship to sequence information disclosed in databases, etc., as long as they have the respective activities.

[0022] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term also encompasses those assembled into complex polypeptide chains. The term also encompasses naturally occurring or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety). This definition also encompasses, for example, polypeptides containing one or more analogs of an amino acid (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" refers collectively to an organic compound containing an amino group and a carboxyl group. When an antibody according to an embodiment of the present disclosure comprises a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Furthermore, any amino acid in the amino acid sequence may form a salt or solvate. Any amino acid in the amino acid sequence may be in the L- or D-form. Even in such cases, the protein according to the embodiment of the present disclosure can be said to contain the above-mentioned "specific amino acid sequence." Known chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.). The amino acids may be natural or unnatural, as long as they satisfy the objectives of the present disclosure.

[0023] As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably and refer to any length of nucleotide polymer, including DNA and RNA.This term also includes "oligonucleotide derivative" or "polynucleotide derivative"."Oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that contains a nucleotide derivative or has an unusual internucleotide bond, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in the oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which the uracil in the oligonucleotide has been substituted with C-5 propynyl uracil, oligonucleotide derivatives in which the uracil in the oligonucleotide has been substituted with C-5 thiazole uracil, oligonucleotide derivatives in which the cytosine in the oligonucleotide has been substituted with C-5 propynyl cytosine, oligonucleotide derivatives in which the cytosine in the oligonucleotide has been substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in the DNA has been substituted with 2'-O-propyl ribose, and oligonucleotide derivatives in which the ribose in the oligonucleotide has been substituted with 2'-methoxyethoxy ribose. Unless otherwise indicated, a particular nucleic acid sequence is also intended to encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" may be natural or non-natural.

[0024] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes. Herein, comparisons of amino acid and nucleotide sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.2.28 (published April 2, 2013). Identity values ​​herein generally refer to values ​​obtained by aligning sequences using the above-mentioned BLAST under default conditions. However, if changing parameters results in a higher value, the highest value is used as the identity value. When identity is evaluated in multiple regions, the highest value among them is used as the identity value. Similarity is a numerical value that takes into account similar amino acids in addition to identity.

[0025] (Preferred embodiment) Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0026] In one aspect of the present disclosure, pyrrole-imidazole polyamides (PIPA) are provided that specifically bind to the binding domain of a protozoan transcription factor.

[0027] Parasitic diseases caused by protozoan parasites such as malaria are one of the world's biggest infectious disease problems, and no fundamental treatment has yet been developed. For example, in the case of malaria infection, antimalarial drugs such as hydroxychloroquine and artemisinin have been developed, but due to the emergence of drug-resistant protozoan parasites, eradication is still a long way off, and the disease remains a pathogen that causes serious infectious diseases.

[0028] For example, malaria parasites possess a highly unique transcriptional machinery regulated exclusively by the Apicomplexa AP2 (ApiAP2) family of transcription factors (TFs). These essential TFs are highly conserved among protozoa in the Apicomplexa phylum, including Plasmodium malaria, Toxoplasma gondii, and Cryptosporidium diffusum. In contrast, in higher eukaryotes, transcription is regulated by a complex mechanism mediated by various transcription factors. Indeed, AP2-O, one of the stage-specific AP2 TFs, regulates the expression of over 500 genes, including at least genes essential for the subsequent morphogenesis. These findings suggest that AP2 TFs could be attractive targets for antimalarial drugs to prevent the development of drug-resistant mutants.

[0029] Pyrrole-imidazole polyamides (PIPAs) are small organic compounds with sequence-specific DNA binding activity that can directly inhibit the transcription of specific target genes. By binding to the appropriate region of a promoter, PIPA inhibits the function of TFs, resulting in the inhibition of target gene transcription. PIPA efficiently translocates into the nucleus both in vitro and in vivo without the need for a drug delivery system (DDS), giving it a significant advantage over other nucleic acid-based gene silencing techniques, such as siRNA, which cannot penetrate the cell membrane. While PIPA can in principle inhibit transcription in all organisms, it has primarily been used to regulate transcription in mammalian cells and has not been used in protozoa. Furthermore, PIPA has primarily been used to specifically inhibit a single target gene, or occasionally multiple genes, and has not been used to function as a transcription factor mimetic.

[0030] In one embodiment of the present disclosure, a PIPA of the present disclosure specifically binds to the binding domain of a protozoan transcription factor. The protozoan is not particularly limited as long as morphological changes in each stage of its life cycle are controlled by a transcription factor, and examples thereof include malaria, Leishmania, Toxoplasma, Cryptosporidium, Coccidia, Babesia, Theileria, and Cystoisospora. In one embodiment, the protozoan may be a protozoan of the phylum Apicomplexa. In one embodiment, a PIPA of the present disclosure that can function as a pseudo-transcription factor can specifically bind to the binding domain of an AP2 family transcription factor. In another embodiment, it is understood that the protozoan targeted by the PIPA of the present disclosure can be any protozoan other than those exemplified above, such as malaria, Leishmania, Toxoplasma, Cryptosporidium, Coccidia, Babesia, Theileria, and Cystoisospora, as long as the transcription factor is an AP2 family transcription factor.

[0031] Malaria parasites are transmitted by Anopheles mosquitoes, and sporozoites, which are salivary gland-infecting parasites, are injected into the host's body and invade hepatocytes. They then divide into merozoites, which are released into the bloodstream and then infect red blood cells. Within red blood cells, merozoites grow and multiply in the following order: ring form, trophozoite, and schizont. Some of these form gametocytes, which enter the mosquito's body via blood feeding, differentiate into oocysts, and new sporozoites are produced within the oocysts. In one embodiment of the present disclosure, the PIPA of the present disclosure can inhibit all morphological changes in each life cycle, and at least inhibits the morphological change into gametocytes. In one embodiment, the PIPA of the present disclosure can inhibit the morphological change into schizonts.

[0032] As shown in the Examples, the present disclosure demonstrates for the first time that PIPAs capable of functioning as pseudo-transcription factors, such as AP2-PIPA, can pass through three membranes: the nuclear membrane of the protozoan, the cell membrane of the protozoan, and the cell membrane of an infected cell (e.g., an erythrocyte), inhibit the function of protozoan transcription factors in intracellular parasites such as malaria, and can be used as therapeutic or preventive agents for diseases caused by protozoan parasites. Furthermore, the present disclosure demonstrates for the first time that PIPAs capable of functioning as pseudo-transcription factors are effective against primitive transcription control mechanisms (i.e., transcription control mechanisms that have only a small number of transcription factors compared to mammals, i.e., one type, a few types, or no transcription factors, or in which specific conserved DNA sequences are commonly important for the control of multiple genes). In one embodiment, the use of a PIPA capable of functioning as a pseudo-transcription factor of the present disclosure prevents the original transcription factor from binding to its binding sequence, thereby inhibiting transcription and its activity associated with binding.

[0033] In other words, the present disclosure provides a unique method for effectively and specifically inhibiting the transcriptional control mechanism in protozoa that parasitize intracellular and extracellular compartments and possess a primitive transcriptional control mechanism. Therefore, the protozoa whose transcription factor function is inhibited by the PIPA of the present disclosure may be any protozoa that possesses a primitive transcriptional control mechanism. Examples of protozoa that possess a primitive transcriptional control mechanism include protozoa belonging to the phylum Apicomplexa, as well as Leishmania, Trypanosoma, Entamoeba histolytica, and Trichomonas. When targeting protozoa whose transcription factors have not yet been identified, the effects of the PIPA of the present disclosure can be achieved by targeting PIPA to a conserved sequence in the promoter that has been revealed by genome analysis or the like.

[0034] Primitive transcriptional regulatory mechanisms in protozoa other than those belonging to the Apicomplexan phylum include the production of long polycystric mRNAs in Leishmania (Journal of Biomedicine and Biotechnology Volume 2010, Article ID 525241, 15 pages). Long polycystric mRNAs are also known to be produced in Trypanosoma (Trends in Parasitology October 2011, Vol. 27, No. 10). In Entamoeba histolytica, although the number of transcription factors is unknown, three consensus sequences have been identified (Front. Microbiol. 10:1921. doi: 10.3389 / fmicb.2019.01921). Furthermore, in Trichomonas, IBP39 is known to regulate over 75% of genes (Molecular Microbiology. 2021;115:959-967). Therefore, by designing PIPA to target specific sequences or factors that function in this primitive transcriptional control mechanism, it is possible to inhibit their function and use it as a therapeutic or preventive agent for diseases caused by protozoa.

[0035] In one embodiment, the PIPA of the present disclosure can specifically bind to the binding domain of a transcription factor specific to a protozoan. In one embodiment, in the case of a PIPA that targets a general transcription factor, the transcription function of the protozoan can be specifically inhibited by a delivery means that specifically delivers the PIPA to the protozoan.

[0036] Dervan et al. demonstrated that synthetic pyrrole-imidazole polyamides (PIPAs) bind to DNA with excellent specificity and very high affinity (Nature 382, ​​559-561 (1996)). DNA recognition by PIPAs depends on the amino acid pairing of imidazole-pyrrole or pyrrole-pyrrole pairs in the minor groove.

[0037] The sequence specificity of PIPA is determined by pairing rules for minor groove-binding polyamides derived from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids: specifically, Py / Im pairs target CG base pairs, Py / Py pairs target AT and TA base pairs, and Im / Py pairs target GC base pairs.

[0038] In one embodiment, the PIPA of the present disclosure binds to dsDNA according to the pairing rules for polyamide subunit recognition of nucleotide bases.More specifically, pyrrole, imidazole, 3-hydroxypyrrole derivatives and aliphatic amino acid residues located in layers form a structure that recognizes specific target nucleotide base pairs in the minor groove of dsDNA.Selected aromatic and aliphatic amino acids are incorporated into polyamide, and these residues remain unpaired with other amino acid residues.The polyamide molecule is crescent-shaped, which can form a complex with the minor groove of double-stranded DNA.

[0039] In one embodiment, two polyamides (PIPAs) can be covalently linked by a turn unit such as γ-aminobutyric acid to increase the binding affinity to the target sequence. Such polyamides are called "hairpin polyamides" because they adopt a hairpin-like structure when complexed with DNA. The imidazole and pyrrole carboxamide arrangement in the polyamide determines the DNA sequence specificity of the ligand according to the carboxamide scheme for recognizing nucleotide pairs. In some cases, one or several pyrrole carboxamide units can be replaced with β-alanine moieties to adjust the curvature of the DNA and polyamide. Polyamides with chiral R2,4-diaminobutyric acid instead of γ-aminobutyric acid as the turn unit can bind to DNA with even higher affinity.

[0040] In one embodiment, the PIPA of the present disclosure can include aliphatic amino acid residues in its structure, and the aliphatic amino acid residues can include molecules having an amino group and a carboxy group. In one embodiment, the aliphatic amino acid residues can include glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid.

[0041] The aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp), can be incorporated into PIPA and paired with the opposing Py to design polyamide DNA-binding ligands that discriminate between A·T and T·A nucleotide pairs. Substituting one hydrogen atom on the pyrrole with a hydroxy group in the Hp / Py pairing restricts the affinity and specificity of the polyamide tenfold. By using Hp with Py and Im in four pairs of aromatic amino acid residues (Im / Py, Py / Im, Hp / Py, and Py / Hp), polyamides can be designed and synthesized that selectively discriminate between all four Watson-Crick base pairs in the minor groove of double-stranded DNA.

[0042] In a preferred embodiment, the present disclosure provides PIPAs having a carboxamide bond that distinguishes between A·T, T·A, C·G, and G·C base pairs in the minor groove of dsDNA. The present disclosure encompasses PIPAs having a γ-aminobutyric acid that forms a hairpin loop with the respective carboxamide pair elements on each end of the PIPA. Preferably, the γ-aminobutyric acid is chiral (R)-2,4-diaminobutyric acid.

[0043] The present disclosure also encompasses PIPAs containing β-alanine substituted for Py, which is normally used in a carboxamide bond pair that pairs with a particular nucleotide pair. β-alanine is represented as β in the formula. β becomes a member of a carboxamide bond pair and functions to optimize hydrogen bonding with the nucleotide pair of the adjacent amino acid moiety. The present disclosure also encompasses the replacement of the β-β bond pair with a non-Hp-containing bond pair. Thus, in addition to Hp / Py and Py / Hp, the bond pairs are Py / Py, Im / Py, Py / Im, Im / β, β / Im, Py / β, β / Py, and β / β.

[0044] In general, the present disclosure provides PIPAs suitable for inhibiting morphological transitions of protozoa into each life cycle, the PIPAs comprising Py and Im structures selected to correspond to the nucleotide sequence of an identified dsDNA target, aliphatic amino acid residues selected from the group consisting of glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid, and optionally a terminal alkylamino residue.

[0045] In one embodiment of the present disclosure, the PIPA of the present disclosure may have a hairpin structure or a cyclic structure according to the above rules, or two linear PIPAs may be used in combination.

[0046] In one embodiment of the present disclosure, the PIPA of the present disclosure comprises at least one aliphatic amino acid residue, which is β-alanine. In a preferred embodiment, the terminal alkylamino residue is an N,N-dimethylaminopropyl residue. The hairpin molecule is formed by aliphatic amino acid residues, such as γ-aminobutyric acid or, more preferably, R2,4-diaminobutyric acid.

[0047] In one embodiment of the present disclosure, the binding region of a protozoan transcription factor to which a PIPA of the present disclosure specifically binds comprises 5'-TGCATG-3' (SEQ ID NO: 1) or a modified sequence thereof, and the modified sequence can include a sequence in which any one base in 5'-TGCATG-3' (SEQ ID NO: 1) is deleted or mutated, or a sequence in which one base is added to any position in 5'-TGCATG-3' (SEQ ID NO: 1). Such binding regions include NGCATG (SEQ ID NO: 2), TNCATG (SEQ ID NO: 3), TGNATG (SEQ ID NO: 4), TGCNTG (SEQ ID NO: 5), TGCANG (SEQ ID NO: 6), and TGCATN (SEQ ID NO: 7), where N is A, T, G, or C.

[0048] In one embodiment of the present disclosure, the binding region of a protozoan transcription factor to which a PIPA of the present disclosure specifically binds comprises 5'-TGCACT-3' (SEQ ID NO: 8) or a modified sequence thereof, and the modified sequence can include a sequence in which any one base in 5'-TGCACT-3' (SEQ ID NO: 8) is deleted or mutated, and a sequence in which one base is added to any position in 5'-TGCACT-3' (SEQ ID NO: 8). Such binding regions include NGCACT (SEQ ID NO: 9), TNCACT (SEQ ID NO: 10), TGNACT (SEQ ID NO: 11), TGCNCT (SEQ ID NO: 12), TGCANT (SEQ ID NO: 13), and TGCACN (SEQ ID NO: 14), where N is A, T, G, or C.

[0049] In one embodiment of the present disclosure, the PIPA of the present disclosure has the following structure: [ka] or [ka] where: L is a C2-6 alkyl linker; R1 and R2 are optionally substituted alkyl, and R1 and R2 together may form a C2-6 alkyl linker; X can be a bond or an aliphatic amino acid residue.

[0050] In one embodiment of the present disclosure, the PIPA of the present disclosure has the following structure: [ka] This can be done (AP2-1).

[0051] In other embodiments, the PIPA of the present disclosure has the following structure: [ka] This can be done (AP2-2).

[0052] In other embodiments, the PIPA of the present disclosure has the following structure: [ka] This can be done (AP2-3).

[0053] In other embodiments, the PIPA of the present disclosure has the following structure: [ka] This can be done (AP2-4).

[0054] In other embodiments, the PIPA of the present disclosure has the following structure: [ka] This can be done (AP2-5).

[0055] In one embodiment of the present disclosure, the PIPA of the present disclosure can have a binding affinity to the binding domain as a dissociation constant (Kd value) of about 500 nM or less. In one embodiment, the PIPA of the present disclosure can have a binding affinity to the binding domain as a dissociation constant (Kd value) of about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, or about 10 nM or less. Since the binding affinity (dissociation constant) of transcription factors of mammalian cells to nucleic acids is approximately 10 nM to several hundred nM, it is expected that protozoan transcription factors will also have a similar binding affinity.

[0056] In one embodiment of the present disclosure, the PIPA of the present disclosure is preferably cell-permeable and can inhibit gene transcription in vivo, in vitro, or in a cell-free system. Such polyamide molecules can be suitably used to inhibit the function of protozoan transcription factors.

[0057] In a related embodiment, one aspect of the present disclosure provides pyrrole-imidazole polyamides (PIPAs) that specifically bind to the binding domain of a protozoan transcription factor for inhibiting the function of the protozoan transcription factor.

[0058] (Use as a pseudo-transcription factor) The PIPA of the present disclosure can also be used as a transcription factor mimetic. Thus, in one aspect of the present disclosure, there is provided a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor for use as a transcription factor mimetic.

[0059] In yet another aspect, there is provided a composition comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor for use as a pseudo-transcription factor.

[0060] In one embodiment, by using a PIPA of the present disclosure that can function as a pseudo-transcription factor, the native transcription factor cannot bind to its binding sequence, thereby inhibiting the transcription and activity associated with binding. Therefore, by making the PIPA of the present disclosure function as a pseudo-transcription factor, it is possible to inhibit the function of a protozoan transcription factor. Therefore, in one aspect of the present disclosure, there is provided a method for inhibiting the function of a protozoan transcription factor in a subject, the method comprising contacting the protozoan transcription factor in the subject with an effective amount of a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding region of the protozoan transcription factor.

[0061] In another aspect of the present disclosure, there is provided a method for using pyrrole imidazole polyamide (PIPA) as a pseudo-transcription factor in a subject, the method comprising applying to the subject an effective amount of PIPA that specifically binds to the binding domain of a protozoan transcription factor.

[0062] (conjugate) In another aspect of the present disclosure, a conjugate is provided, comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor and a protozoan-specific factor different from the PIPA. As described herein, the PIPA of the present disclosure can specifically bind to the binding domain of a protozoan transcription factor and inhibit its transcription. By creating a conjugate between this PIPA and a factor that specifically binds to a protozoan (protozoan-specific factor), a drug delivery system for PIPA can be constructed.

[0063] In one embodiment, the protozoan-specific factor for preparing the conjugate of the present disclosure may directly act on the protozoan, or may be a substance that can be expected to indirectly provide some therapeutic effect, suppress symptoms, and / or prevent protozoan infection, even if it does not directly act on the protozoan. For example, it may be a substance that activates immune cells or protects red blood cells, and the like. Factors that can be bound to the PIPA of the present disclosure to form the conjugate preferably have a low molecular weight (approximately 500 to approximately 2000), and more preferably have a molecular weight that does not affect the delivery efficiency of PIPA, for example, approximately 500 to approximately 1000. By forming a conjugate with such a factor and PIPA, the conjugate of the present disclosure can directly or indirectly provide some therapeutic effect, suppress symptoms, and / or preventive effect against protozoan infection. In one embodiment, the protozoan-specific factor for preparing the conjugate of the present disclosure may be one that specifically binds to the target protozoan, preferably one that specifically binds to the target protozoan and functions as an inhibitor. For example, a protozoan-specific factor can be one that binds to a surface protein on malaria-infected red blood cells. Such factors include pyridazinone derivatives, and examples of pyridazinone derivatives include MBX-4055 represented by the following formula and its derivatives.

[0064] JPEG0007756380000032.jpg51127 JPEG0007756380000033.jpg48127 JPEG0007756380000034.jpg44127 JPEG0007756380000035.jpg41127

[0065] MBX-4055 is known to inhibit the growth of malaria. Specifically, malaria proteins expressed on the surface of red blood cells are necessary for the uptake of components necessary for malaria growth from outside the red blood cells, and MBX-4055 inhibits this function, thereby inhibiting the growth of malaria. In one embodiment of the present disclosure, focusing on the function of MBX-4055, which binds to malaria proteins expressed on infected red blood cells, MBX-4055 can be used as a drug delivery system, and a synergistic effect with the inhibitory action of PIPA can be expected.

[0066] In another embodiment, the protozoan-specific factor may be a factor that inhibits the growth of malaria, leishmania, toxoplasma, cryptosporidium, and / or coccidia. Such factors include, for example, amphotericin B, which binds to a cell surface protein of infected leishmania (Life Sciences, Volume 322, June 1, 2023, 121314). In another embodiment, the protozoan-specific factor may be an inhibitor of toxoplasma, such as the compound described in Microorganisms 2021, 9(9), 1960. In another embodiment, the protozoan-specific factor may be an inhibitor of Cryptosporidium, such as the compound described in Animal Diseases, Volume 1, Article Number: 3 (2021). In another embodiment, the protozoan-specific factor may be an inhibitor of coccidia, such as the compound described in Journal of the Japanese Society of Veterinary Medicine, Vol. 71, pp. 166-169 (2018).

[0067] In one embodiment, the PIPA and the protozoan-specific factor of the present disclosure may be directly linked to form a conjugate, or may be linked via a linker. Any linker may be used as the linker, as long as it can function as the conjugate of the present disclosure, for example, as a drug delivery system, and examples of such linkers include C1-C6 alkyl linkers.

[0068] In yet another aspect, there is provided a composition for inhibiting the function of a protozoan transcription factor, the composition comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of the protozoan transcription factor.

[0069] In yet another aspect, there is provided a protozoan transcription factor inhibitor comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor.

[0070] In one aspect of the present disclosure, there is provided a therapeutic or preventive agent for a disease caused by a protozoan, comprising a protozoan transcription factor inhibitor, wherein the protozoan transcription factor inhibitor comprises a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of the protozoan transcription factor.

[0071] In one aspect of the present disclosure, there is provided a pyrrole-imidazole polyamide (PIPA) that specifically binds to the binding domain of a protozoan transcription factor for treating or preventing a disease caused by a protozoan.

[0072] In one aspect of the present disclosure, there is provided a method for producing a protozoan transcription factor inhibitor comprising a pyrrole-imidazole polyamide (PIPA), the method comprising the steps of providing a binding domain of a protozoan transcription factor and designing PIPA to specifically bind to the binding domain. In one embodiment, the PIPA and the protozoan may be those described elsewhere herein.

[0073] In one embodiment, the designing step can include linking pyrroles and / or imidazoles selected to correspond to the nucleotide sequence of the binding region, and, if necessary, substituting β-alanine for one or more pyrroles or imidazoles in the linked pyrroles and / or imidazole molecules. In this case, the design and synthesis of PIPAs are preferably performed in accordance with the rules for PIPA structure as described above.

[0074] In one aspect of the present disclosure, there is provided a method for treating or preventing a disease caused by a protozoan in a subject, the method comprising administering to the subject an effective amount of a protozoan transcription factor inhibitor, the protozoan transcription factor inhibitor comprising a pyrrole-imidazole polyamide (PIPA) that specifically binds to a binding domain of the protozoan transcription factor. In one embodiment, the PIPA and the protozoan may be those described elsewhere herein.

[0075] The PIPA of the present disclosure, as well as its pharmaceutically acceptable salts, can be formulated into pharmaceutical or therapeutic compositions, formulations, or preparations.The pharmaceutically acceptable salts of the PIPA of the present disclosure are formed by methods known in the art using strong or moderate, non-toxic, organic or inorganic acids or bases, as appropriate.Examples of salts included in the present disclosure include maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate, and nitrate.

[0076] The PIPA of the present disclosure has the ability to treat or prevent diseases caused by protozoa. The compositions of the present disclosure may be active themselves or may act as prodrugs that are converted to active forms in vivo.

[0077] The PIPA of the present invention and its pharmaceutically acceptable salts can be incorporated into conventional dosage forms such as capsules, tablets, or injectable preparations.Solid or liquid pharmaceutically acceptable carriers can be used.Pharmaceutical compositions designed for delayed release can also be formulated.

[0078] Preferably, PIPA of the present disclosure is administered systemically, for example by injection.When used, injection can be by any known route, preferably intravenous, subcutaneous, intramuscular, intracranial or intraperitoneal.Injectable preparation can be prepared into known forms such as solution or suspension, solid form suitable for dissolving or suspending in liquid before injection, or emulsion.

[0079] Pharmaceutical preparations are prepared according to conventional techniques of pharmaceutical chemistry, including processes such as mixing, granulating, and compressing, if necessary for tablet form, or mixing, filling, and dissolving the ingredients, as appropriate, to obtain the desired product for oral or parenteral administration, including topical, transdermal, intravaginal, intranasal, intrabronchial, intracranial, intraocular, intraotic, and rectal administration. Pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0080] Although the preferred route of administration is systemic administration, the pharmaceutical composition can also be administered topically or transdermally (e.g., as an ointment, cream, or gel), orally, rectally (e.g., as a suppository), parenterally, continuously by injection or infusion, intravaginally, intranasally, intrabronchially, intracranially, intraauricularly, or intraocularly. In one embodiment, in the case of PIPA, which targets a general transcription factor, the transcriptional function of the protozoan can be specifically inhibited by a delivery means that delivers the PIPA specifically to the protozoan.

[0081] The PIPA-containing compositions of the present disclosure can also be administered in combination with one or more additional compounds used to treat a disease or condition.

[0082] An effective amount of PIPA for treating a disease or condition can be determined using recognized in vitro systems or in vivo animal models for the particular disease or condition.

[0083] The therapeutic method of the present disclosure includes administering an effective amount of a protozoan transcription factor inhibitor. The PIPA-containing formulation of the present disclosure can be administered systemically or locally, either alone or as a mixture of components. Administration routes include topical, intravenous, oral, or implant administration. For example, PIPA can be administered by means including, but not limited to, topical formulations, intravenous injection or infusion, oral ingestion, or local administration in the form of intradermal injection or implant. Additional administration routes include subcutaneous, intramuscular, or intraperitoneal injection of the PIPA of the present disclosure in a conventional or convenient form. Liposomal or lipophilic formulations can also be used as desired. For topical administration, the polyamide can be formulated into standard topical formulations and compositions, including lotions, suspensions, or pastes. Where PIPA can be readily applied to target cells or tissues via the oral route, oral administration of the appropriate formulation is also appropriate.

[0084] The dosage of PIPA can be optimized by one of ordinary skill in the art depending on factors including, but not limited to, the PIPA selected, the physical delivery system in which it is delivered, the patient, and the judgment of a skilled practitioner.

[0085] (General technology) The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and can be found in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). These methods are described in PCR Applications: Protocols for Functional Genomics, Academic Press, and in Special Edition of Experimental Medicine "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, and the relevant portions (possibly in their entirety) of these methods are incorporated herein by reference.

[0086] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used. Other examples include Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0087] Other publications related to PIPA include: Jordan L. Meier et al., J. Am. Chem. Soc. 2012, 134, 17814-17822; Journal of the American Chemical Society, 2012, vol. 134, pp. 17814-17822; Nature, 1998, vol. 391, p. 468; Advanced Drug Delivery Reviews, 2019, vol. 147, pp. 66-85; Bull. Chem. Soc., 2020, vol. 93, pp. 205-215.

[0088] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within a range" of "two values" is specified, the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0089] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0090] reagent Pyrrole-imidazole polyamides were synthesized at the Peptide Institute, Inc. (Osaka, Japan). Lipopolysaccharide was purchased from Invivogen (Carlsbad, CA, USA; tlrl-eblps).

[0091] cell Raw246.7, NIH3T3, U937, and A549 cells were obtained from ATCC. Red blood cells were obtained from whole blood as described below. Human blood was provided by healthy volunteers in accordance with the guidelines of the University of Tokyo or Osaka University.

[0092] qRT-PCR analysis Total RNA used for in vitro stimulation or in vivo injection was extracted from cells using NucleoSpin RNA (TAKARA BIO, Shiga, Japan). DNA primers were obtained from FASMAC (Kanagawa, Japan).

[0093] Example 1: Transcription inhibitors against P. falciparum In this example, an example of a transcription inhibitor against P. falciparum is shown. To develop a transcription inhibitor for P. falciparum, we first determined the target transcription factor and its sequence. Since one of the most notable toxic effects of the malaria parasite is the destruction of red blood cells, we decided to inhibit the formation of merozoites, which destroy cells at the final stage of the blood stage and cause new red blood cell invasion. Malaria parasites form schizonts immediately before merozoite formation. AP2-Sc is expressed in schizonts and plays an important role as an essential transcription factor.

[0094] We then selected AP2-Sc as a target and performed ChIP-seq analysis to comprehensively identify its binding sequences. The first hit was a sequence called TGCATG (SEQ ID NO: 1), which accounted for approximately 50% of the precipitated DNA segments. TGCATG (SEQ ID NO: 1) was specifically located in the promoter regions of schizont-specific genes, such as AMA1 (PF3D7_1133400) and GAMA (PF3D7_0828800). However, no other developmental stage-specific genes, such as CTRP, were detected. These results suggest that TGCATG (SEQ ID NO: 1) may be important for schizont formation in P. falciparum.

[0095] Based on ChIPC-seq analysis, we then constructed two PIPAs, AP2-PIPA1 and AP2-PIPA2, targeting TGCATG (SEQ ID NO: 1) and TGCACT (SEQ ID NO: 8), respectively, as controls. As shown in Figure 1a, AP2-PIPA is a hairpin-type PIPA containing β-alanine in its structure, with total molecular weights of 1423 and 1351, respectively.

[0096] Next, we confirmed the binding affinity of PIPA to dsDNA containing the AP2-PIPA target sequence (Fig. 1b). As designed, AP2-PIPA bound to the target sequence with high avidity, with a Kd value of 4.0 nM (Fig. 1c). Consistent with previous reports demonstrating low off-target toxicity of PIPA, AP2-PIPA showed no toxicity in either mouse cell lines (Raw264.7 cells, NIH3T3 cells) or human cell lines (U937 cells, A549 cells) (Fig. 2a and 2b). These analytical results support the safety of PIPA, at least in vitro, within this dose range.

[0097] Furthermore, we investigated the toxicity of a single intraperitoneal administration of AP2-PIPA1 in mice. The doses were 0 (PBS), 5, 10, and 20 mg / kg, and the animals were observed for 7 days after administration. Toxicity assessment included viability and general observation, weight measurement, hematology, blood chemistry, and necropsy. No deaths were observed, and no abnormal changes were observed in general observation, weight change, hematology, blood chemistry, or necropsy. Significant increases in AST activity were observed in the 5 and 20 mg / kg groups, and although not significantly different, there was a tendency for an increase in AST activity in the 10 mg / kg group (Figure 5a). However, individual values ​​in these groups were within the background range (34–58 IU / L), suggesting this was due to physiological fluctuations. Based on these results, we estimate that the minimum lethal dose of AP2-PIPA1 following a single intraperitoneal administration exceeds 20 mg / kg.

[0098] Furthermore, the toxicity of multiple intravenous administration of AP2-PIPA1 was examined using mice. Mice were administered various doses of AP2-PIPA1 orally or intraperitoneally once daily for 7 days. Blood was collected on day 8, and AST and ALT were measured (Figure 5b). Significance tests were performed on the 3 mg / kg and 10 mg / kg groups compared to the control group for each administration route. Bartlett's test for equal variances indicated a p>0.01, and Dunnett's two-tailed test indicated no significant difference, with a p>0.05. These results suggest that AP2-PIPA1 does not exhibit significant toxicity within the dose range examined in this study.

[0099] We next examined the effect of AP2-PIPA on the in vitro growth of the blood-stage P. falciparum 3D7 laboratory strain. Human erythrocytes were infected with P. falciparum and cultured in the presence or absence of AP2-PIPA. At 24 and 48 hours postinfection, microscopic observation with Giemsa staining and quantitative counting of parasites and their stages were performed by FACS analysis. As shown in Figure 3a, AP2-PIPA1 arrested the P. falciparum life cycle at the trophozoite stage in a dose-dependent manner compared with AP2-PIPA2. Furthermore, FACS analysis confirmed that P. falciparum cultured in the presence of AP2-PIPA1 exhibited morphological arrest at the trophozoite stage at both 24 and 48 hours postinfection (Figures 3b and 3c). These results suggest that AP2-PIPA1 specifically inhibits the transition to the schizont stage without affecting trophozoite formation, consistent with the role of the target sequence binding to AP2-Sc. AP2-PIPA2 was thought to be effective at a higher concentration range than the concentrations used in this experiment.

[0100] Furthermore, we investigated the effect of AP2-PIPA on the growth of artemisinin-resistant strains in vitro (Fig. 4). Human erythrocytes were infected with each malaria strain and cultured in the presence or absence of various concentrations of AP2-PIPA. Parasitemia was assessed 72 hours after infection, and the growth rate of malaria treated with each concentration was examined. From the results, the IC value of AP2-PIPA for each malaria strain was determined. 50 As a result, AP2-PIPA1 showed a lower IC 50 In contrast, AP2-PIPA2 did not exhibit any inhibitory effect on any of the malaria strains, suggesting that it is effective at a higher concentration range than the concentrations used in this experiment.

[0101] We further verified the effect of AP2-PIPA1 in mouse infection experiments. Mice were infected with a mouse malaria strain and intraperitoneally administered AP2-PIPA1 at the time points indicated in the upper diagram of Figure 6 (days 1, 2, 3, 4, 5, 6, and 7). Blood samples were collected 11 or 13 days after infection, and parasitemia was assessed. A dose-dependent suppression of parasitemia was observed. Higher doses, particularly those at 30 mg / kg, suppressed parasitemia and resulted in mouse death (see the bottom graph of Figure 6). These results confirmed the antimalarial effect of AP2-PIPA1 in mice. Furthermore, multiple administration of the high dose of 30 mg / kg is likely to result in toxicity.

[0102] In this study, we developed a novel PIPA-based inhibitor targeting the consensus binding sequence of AP2-Sc, an AP2 family TF essential for P. falciparum schizont formation. Unlike conventional strategies that rely on promoter analysis to identify PIPA target sequences, we focused on the AP2-Sc binding sequence identified by chromatin immunoprecipitation sequencing (ChIP-seq) analysis, because AP2 TFs bind to cis-elements in multiple target genes. This strategy allows AP2-Sc-targeting PIPA (AP2-PIPA) to compete with AP2-Sc for all genes with consensus cis-elements in their promoters, rather than just specific genes. Indeed, AP2-PIPA was confirmed to potently inhibit P. falciparum schizont formation in in vitro culture. This demonstrated, for the first time, that PIPA inhibits the proliferation and life cycle of the protozoan parasite.

[0103] All genes with consensus cis-elements in their promoters can compete with AP2-Sc, including ARNP (PF3D7_0511600), MSP7 (PF3D7_1335100), MSP9 (PF3D7_1228600), and EXP1 (PF3D7_1121600).

[0104] Example 2: Transcription factor inhibition using PIPA in Leishmania It is known that the primitive transcriptional control mechanism of Leishmania is the production of long polycystic mRNAs (Journal of Biomedicine and Biotechnology Volume 1, 2019). (2010, Article ID 525241, 15 pages). Because multiple proteins are produced from this specific mRNA, we designed a PIPA that inhibits the transcription of this polycystic mRNA. The design method was the same as in Example 1.

[0105] The binding affinity of PIPA to the target sequence was confirmed in the same manner as in Example 1, and PIPA that was confirmed to bind to the target sequence with high binding activity was administered to a subject. The inhibitory effect on transcription function in Leishmania was confirmed. The inhibitory effect and mouse infection experiments were performed in the same manner as in Example 1. This confirmed the PIPA and its concentration that are effective in treating Leishmania infection.

[0106] Example 3: Transcription factor inhibition using PIPA in Toxoplasma gondii AP2-PIPA1 is thought to inhibit Toxoplasma gondii, which belongs to the same phylum as the malaria parasite, the Apicomplexan. This is supported by the fact that AP2 transcription factors are conserved within the Apicomplexan phylum, and that the DNA-binding domains of these AP2 transcription factors are conserved, i.e., the binding DNA sequences are thought to be conserved. Indeed, as shown in Figure 4 of THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 43, pp. 31127-31138 and Figure 1 of PNAS June 17, 2008 vol. 105 no. 248393, one of the AP2 transcription factors in Toxoplasma gondii, TgAP2XI-5, is an important transcription factor involved in the expression of over 300 genes. The AP2 DNA-binding domain shows high homology to that of the AP2 transcription factor in malaria parasites. Furthermore, as shown in Figures 4 and 6 of the above-mentioned document, the most likely binding DNA sequence of TgAP2XI-5 is predicted to be GCTAGC, which has only a single-base mismatch with the AP2-PIPA1-inhibitable sequence (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT). To be precise, if the sequence on the 5' side of GCTAGC (SEQ ID NO: 16) is A or T, it will be a perfect match. In other words, there is a 50% probability of a perfect match, and even in the remaining 50% mismatch, AP2-PIPA1 is thought to bind, although the binding strength may be reduced due to the single-base mismatch. Furthermore, as shown in Figures 4 and 6 of the above-mentioned document, the DNA sequence (agctag) (SEQ ID NO: 17) that TgAP2XI-5 can bind to at the fourth position in Figure 4 and the third position in Figure 6 is a perfect match with the AP2-PIPA1-inhibitable sequence (WGCWWG) (SEQ ID NO: 15).Another study has also reported that TGCATGCA (SEQ ID NO: 18) is one of the most likely sequences recognized by AP2 transcription factors from the Apicomplexan phylum, which includes malaria, Toxoplasma, Cryptosporidium, and Coccidia (e.g., Pathogens 2019, 8, 47; doi:10.3390 / pathogens8020047) (Genome Res. 2007 17:311-319), and this sequence contains a 100% match to the AP2-PIPA1-inhibitable sequence (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT)).

[0107] Based on the above, the transcription factor inhibitory effect in Toxoplasma was confirmed using AP2-PIPA1 designed in the same manner as in Example 1. The inhibitory effect and mouse infection experiments were performed in the same manner as in Example 1. This confirmed the concentration of AP2-PIPA1 that is effective in treating Toxoplasma infection.

[0108] Example 4: Transcription factor inhibition using PIPA in Cryptosporidium AP2-PIPA1 is expected to inhibit Cryptosporidium, which belongs to the same phylum as the malaria parasite, the Apicomplexan. This is based on the fact that AP2 transcription factors are conserved in the Apicomplexan phylum, and that the DNA-binding domains of AP2 transcription factors are conserved, i.e., the binding DNA sequences are thought to be conserved. In fact, as shown in Figure 1 of PNAS June 17, 2008, vol. 105, no. 248393, the AP2 DNA-binding domain of Cryptosporidium AP2 shows high homology to that of the AP2 transcription factor of the malaria parasite. Furthermore, the Cryptosporidium AP2 binding DNA sequences shown as Cad8_3230, Cgd1_3520, and Cgd2_3490 in Figure 3 of Nucleic Acids Research, 2014, Vol. 42, No. 13, contain a sequence that perfectly matches the AP2-PIPA1-inhibitable sequence (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT). Furthermore, the most likely Cryptosporidium AP2 binding DNA sequence shown in Figure 5 of the same document contains a sequence that perfectly matches the AP2-PIPA1-inhibitable sequence (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT). Similar findings were reported in PNAS, June 17, 2008, Vol. 105, no. 248393. Another paper also reported that one of the most likely sequences recognized by AP2 transcription factors of the Apicomplexan phylum, which includes malaria, Toxoplasma, Cryptosporidium, and Coccidia, is TGCATGCA (SEQ ID NO: 18) (Pathogens 2019, 8, 47; doi:10.3390 / pathogens8020047) (Genome Res. 2007 17: 311-319), and this sequence contains a 100% match with the sequence that can be inhibited by AP2-PIPA1 (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT)). There is.

[0109] Based on the above, the transcription factor inhibitory effect on Cryptosporidium was confirmed using AP2-PIPA1 designed in the same manner as in Example 1. The inhibitory effect and mouse infection experiments were performed in the same manner as in Example 1. This confirmed the concentration of AP2-PIPA1 that is effective in treating Cryptosporidium infection.

[0110] Example 5: Transcription Factor Inhibition with PIPA in Coccidia AP2-PIPA1 is expected to inhibit Coccidia, which belong to the same phylum as the malaria parasite, Apicomplexa, because, for example, AP2 transcription factors are conserved in the Apicomplexa phylum and the DNA-binding domains of these AP2 transcription factors are thought to be conserved, i.e., the binding DNA sequences are thought to be conserved. It has been shown that the AP2 transcription factors of malaria, Toxoplasma, and Cryptosporidium, which belong to the same phylum Apicomplexan, all bind to a sequence (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT)) that can be inhibited by AP2-PIPA1, and it is thought that the AP2 of Coccidia also binds to a sequence that can be inhibited by AP2-PIPA1. In fact, one of the most likely sequences recognized by the AP2 transcription factor of the Apicomplexan phylum, which includes malaria, Toxoplasma, Cryptosporidium, and Coccidia, is TGCATGCA (SEQ ID NO: 18) (Pathogens 2019, 8, 47; doi:10.3390 / pathogens8020047) (Genome Res. 2007 17: 311-319), and this sequence contains a sequence that is 100% identical to the AP2-PIPA1 inhibitory sequence (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT).

[0111] Based on the above, the transcription factor inhibitory effect on coccidia was confirmed using AP2-PIPA1 designed in the same manner as in Example 1. The inhibitory effect and mouse infection experiments were performed in the same manner as in Example 1. This allowed us to confirm the concentration of AP2-PIPA1 that is effective in treating coccidia infection.

[0112] Example 6: Transcription factor inhibition using PIPA in Babesia AP2-PIPA1 is expected to inhibit Babesia, which belongs to the same phylum Apicomplexa as the malaria parasite. The basis for this is that AP2 transcription factors are conserved in the Apicomplexa phylum, and the DNA binding domains of these AP2 transcription factors are conserved, meaning that the binding DNA sequences are thought to be conserved. In fact, PLOS Neglected Tropical Diseases, DOI:10.1371 / journal.pntd.0004983 November As shown in Figure 4 in PNAS June 17, 2008 vol. 105 no. 248393, Figure 1 in PLOS Neglected Tropical Diseases | DOI:10.1371 / journal.pntd.0003933 August 14, 2015, Figure 3, the AP2 DNA-binding domain of Babesia AP2 binds to the malaria parasite. It shows high homology to the AP2 transcription factor of Plasmodium parasites. In addition, it has been shown that the AP2 transcription factors of Plasmodium malariae, Toxoplasma gondii, and Cryptosporidium, which belong to the same Apicomplexan phylum, all bind to a sequence that can be inhibited by AP2-PIPA1 (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT)). Considering this, together with the conservation of the Babesia AP2 domain, it is thought that Babesia AP2 also binds to a sequence that can be inhibited by AP2-PIPA1.

[0113] Based on the above, the transcription factor inhibitory effect in Babesia was confirmed using AP2-PIPA1 designed in the same manner as in Example 1. The inhibitory effect and mouse infection experiments were performed in the same manner as in Example 1. This allowed us to confirm the concentration of AP2-PIPA1 that is effective in treating babesiosis.

[0114] Example 7: Transcription factor inhibition using PIPA in Theileria AP2-PIPA1 is expected to inhibit Theileria, which belongs to the same phylum Apicomplexa as the malaria parasite. The basis for this is that AP2 transcription factors are conserved in the Apicomplexa phylum, and the DNA-binding domains of these AP2 transcription factors are conserved, meaning that the binding DNA sequences are thought to be conserved. In fact, Figure 4 in PLOS Neglected Tropical Diseases | DOI:10.1371 / journal.pntd.0004983 November 10, 2016, Figure 1 in PNAS June 17, 2008 vol. 105 no. 248393, and Figure 2 in PLOS Neglected Tropical Diseases | DOI:10.1371 / journal.pntd.0003933 August 14, As shown in Figure 3 of 2015, the AP2 DNA-binding domain of Theileria AP2 shows high homology to that of the AP2 transcription factor of Plasmodium falciparum. Furthermore, it has been shown that the AP2 transcription factors of Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium, which all belong to the same Apicomplexan phylum, bind to a sequence that can be inhibited by AP2-PIPA1 (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT)). Considering this, along with the conservation of the Theileria AP2 domain, it is likely that Theileria AP2 also binds to a sequence that can be inhibited by AP2-PIPA1.

[0115] Based on the above, the transcription factor inhibitory effect in Theileria will be confirmed using AP2-PIPA1 designed in the same manner as in Example 1. The inhibitory effect and mouse infection experiments will be performed in the same manner as in Example 1. This will allow us to confirm the concentration of AP2-PIPA1 that is effective in treating equine piroplasmosis.

[0116] Example 8: Transcription factor inhibition using PIPA in other Apicomplexan protozoa It is thought that all AP2 transcription factors of protozoan parasites belonging to the Apicomplexan phylum (such as Cystoisospora) bind to a sequence that can be inhibited by AP2-PIPA1 (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT). In other words, AP2-PIPA1 can exert an inhibitory effect common to the Apicomplexan phylum. In fact, one of the most likely sequences recognized by AP2 transcription factors of the Apicomplexan phylum, which includes malaria, Toxoplasma, Cryptosporidium, and Coccidia, is TGCATGCA (Pathogens 2019, 8, 47; doi:10.3390 / pathogens8020047), which indicates a high degree of commonality. This sequence contains a 100% identical sequence to the AP2-PIPA1 inhibitory sequence (WGCWWG (SEQ ID NO: 15) (W can be either AT or AT).

[0117] Based on the above, AP2-PIPA1 designed in the same manner as in Example 1 will be used to confirm the transcription factor inhibitory effect on other Apicomplexan protozoa (such as Cystoisospora). Inhibitory effects and mouse infection experiments will be performed in the same manner as in Example 1. This will confirm the concentration of AP2-PIPA1 that is effective in treating infections caused by other Apicomplexan protozoa (such as Cystoisospora).

[0118] Example 9: Examples of alternative PIPA structures in malaria Other PIPAs that bind to the same target sequence (AP2) as in Example 1 were designed. These are shown in Figures 7a to 7c. These PIPAs were used to confirm the inhibitory effect on transcription factors of protozoa such as malaria, in the same manner as in Example 1. The inhibitory effect and mouse infection experiments were performed in the same manner as in Example 1. This allowed us to confirm the concentration of AP2-PIPA1 that is effective in treating protozoan infections such as malaria.

[0119] Example 10: Design of PIPAs targeting other malaria target sequences ChIP-seq analysis was performed to search for binding sequences of malaria transcription factors, and TGCACA (SEQ ID NO: 19) was identified. PIPA was designed as in Example 1, and mouse infection experiments were performed to confirm the transcription factor inhibitory effect of PIPA targeting TGCACA (SEQ ID NO: 19).

[0120] Example 11: Preparation of anti-malarial drug Due to its high cell delivery efficiency, PIPA is considered a drug discovery modality that does not require a DDS. Therefore, when AP2-PIPA1 is formulated, it is formulated in the same way as a general drug.

[0121] Examples of common formulations include oral preparations such as pills, capsules, granules, powders, and liquids, topical preparations such as ointments, patches, and lotions, and injectable preparations such as liquids, as well as eye drops, nasal drops, suppositories, inhalants, etc. AP2-PIPA1 is mixed into such formulations and administered to patients by commonly used methods.

[0122] Example 12: In vivo testing The inhibitory effect in vivo was confirmed using AP2-PIPA1 designed in the same manner as in Example 1. Mice transfected with human red blood cells were infected with artemisinin-resistant malaria parasites, and the therapeutic effect in infected mice was confirmed. The effect was confirmed when PIPA was administered alone and when it was administered in combination with artemisinin.

[0123] Humanized mice were generated by injecting NOG-SCID mice with human red blood cells (RBCs). One ml of 50% HCT, A+ve RBCs prepared in 0.5% Albumax and 3.1 mM hypoxanthine was injected into NOG-SCID mice once daily for 3 weeks until 80% human RBCs were established. Subsequently, humanized mice were inoculated with 4 × 10 artemisinin-resistant Plasmodium falciparum (Pf lek122) parasites. 7Mice were infected with synchronized ring-stage parasites, and parasitemia was assessed by determining the percentage of Giemsa-stained positive RBCs. On days 21, 22, 23, 24, and 25, AP2-PIPA1 (AP2-1), artemisinin, or both were injected i.p. at the indicated doses.

[0124] The results are shown in Figure 8. As can be seen from this figure, although the effect of artemisinin could not be confirmed, a therapeutic effect on malaria susceptibility was confirmed in both the administration of PIPA alone and in combination with artemisinin.

[0125] (Example 13: Drug delivery test using conjugates) A conjugate will be created by combining AP2-PIPA1 designed in the same manner as in Example 1 with a compound that specifically binds to a protozoan protein, and the drug delivery effect of this conjugate will be confirmed.

[0126] MBX-4055 and its derivatives, which bind specifically to proteins on the surface of malaria-infected red blood cells (expressed early in infection), will be used as compounds that specifically bind to parasite proteins (Figure 9). Conjugates of AP2-PIPA1 and MBX-4055, as well as conjugates of AP2-PIPA1 and MBX-4055 derivatives, will be created, and the drug delivery effects, transcription factor inhibitory effects, and therapeutic effects of the conjugates will be confirmed.

[0127] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the claims. It is understood that the patents, patent applications, and other documents cited in this specification should be incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth in this specification. This application claims priority to Japanese Patent Application No. 2022-67631 filed with the Japan Patent Office on April 15, 2022, the contents of which are incorporated by reference in their entirety as if they constitute the contents of this application. [Industrial Applicability]

[0128] According to the present disclosure, pyrrole-imidazole polyamide (PIPA) can be used to provide antiprotozoan drugs that target protozoan transcription factors, which will enable drug development for protozoan infections for which no fundamental therapeutic drugs have yet been developed, and is therefore expected to be applied in the medical field. [Sequence List Free Text]

[0129] SEQ ID NOs: 1 to 7: Binding domains of protozoan transcription factors to which PIPA specifically binds according to an embodiment of the present disclosure SEQ ID NOs: 8 to 14: Binding domains of protozoan transcription factors to which PIPA specifically binds according to other embodiments of the present disclosure SEQ ID NOs: 15-23: Target and binding sequences of PIPA according to one embodiment

Claims

1. The following structure: 【Chemistry 3】 Pyrrole-imidazole polyamide (PIPA).

2. The following structure: 【Chemistry 4】 Pyrrole-imidazole polyamide (PIPA).

3. A composition comprising the PIPA of claim 1 or 2 for inhibiting the function of a protozoan transcription factor.

4. A protozoan transcription factor inhibitor comprising the PIPA described in claim 1 or 2.

5. A therapeutic or preventive agent for a disease caused by protozoa, comprising a protozoan transcription factor inhibitor, wherein the protozoan transcription factor inhibitor comprises the PIPA described in claim 1 or 2.

6. A conjugate comprising the PIPA according to claim 1 or 2 and a protozoan-specific factor different from the PIPA, The protozoan-specific factor is MBX-4055 represented by the following formula: 【Chemical 21】 ,or Is it an MBX-4055 derivative represented by the following formula? 【Chemical 22】 ,or Is it an MBX-4055 derivative represented by the following formula? 【Chemical 23】 ,or It is an MBX-4055 derivative represented by the following formula: 【Chemistry 24】 Conjugates.

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