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CN-121986172-A - Method for producing Nucleoside Triphosphates (NTPs)

CN121986172ACN 121986172 ACN121986172 ACN 121986172ACN-121986172-A

Abstract

The present invention relates to novel enzymatic methods for producing Nucleoside Triphosphates (NTPs), particularly modified NTPs, using polyphosphate kinase 2. NTPs produced by the methods of the invention may be used in methods of producing polynucleotides (including oligonucleotides) for treatment.

Inventors

  • J. Horsford
  • D. Foster

Assignees

  • 葛兰素史密斯克莱知识产权发展有限公司

Dates

Publication Date
20260505
Application Date
20240920
Priority Date
20230922

Claims (20)

  1. 1. Use of a polyphosphate kinase (PPK) in the production of sugar-modified and/or alpha-phosphate modified nucleoside triphosphates produced from a corresponding modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate.
  2. 2. The use of claim 1, wherein the PPK is PPK2.
  3. 3. The use of claim 1 or claim 2, wherein the PPK is PPK2-III.
  4. 4. The use as claimed in any one of claims 1 to 3, wherein the PPK has a sequence which is at least 90% identical to SEQ ID No. 22.
  5. 5. The use as claimed in any one of claims 1 to 4, wherein the PPK has the sequence of SEQ ID No. 22.
  6. 6. A method of screening a set of putative and known PPKs to identify an enzyme capable of producing a corresponding modified nucleoside triphosphate using a sugar-modified and/or alpha-phosphate modified nucleoside monophosphate or nucleoside diphosphate as a substrate, the method comprising: a) Identifying sequences encoding the set of putative and known PPKs; b) Synthesizing the PPK; c) Purifying the PPK; d) Mixing each of said purified PPKs with said sugar-modified and/or alpha-phosphate-modified nucleoside monophosphates or nucleoside biphosphates and polyphosphates to form a reaction mixture, and E) Analyzing the presence of the corresponding modified nucleoside triphosphates in each of the reaction mixtures to determine which enzyme or enzymes produce the corresponding modified nucleoside triphosphates.
  7. 7. The method of claim 6, wherein the sequences in step a) are identified using a sequence database and bioinformatics tools.
  8. 8. The method of claim 6 or claim 7, wherein the sequence in step a) encodes a protein between 150 and 600 amino acid residues in length.
  9. 9. The method of any one of claims 6 to 8, wherein the PPK is synthesized in step b) to have an N-terminal hexahistidine tag and purified in step c) using nickel nitrilotriacetate.
  10. 10. The method of any one of claims 6 to 9, wherein step d) comprises incubating the sugar-modified and/or alpha-phosphate-modified nucleoside mono-or diphosphate, polyphosphate and PPK at 25 ℃ for 24 hours and quenching the reaction.
  11. 11. The method of claim 10, wherein the reaction is quenched with EDTA for 15 minutes at 80 ℃.
  12. 12. The method of any one of claims 6 to 11, wherein the reaction mixture is centrifuged and the supernatant is analyzed for the presence of the corresponding modified nucleoside triphosphate.
  13. 13. The method of claim 12, wherein the reaction mixture is centrifuged at 4000 g minutes.
  14. 14. The method of claim 12 or claim 13, wherein the supernatant is analyzed using HPLC or UPLC.
  15. 15. The method of any one of claims 6 to 14, wherein the enzyme capable of producing a corresponding modified nucleoside triphosphate is further engineered to increase the percent (%) conversion to the corresponding modified nucleoside triphosphate.
  16. 16. An enzymatic method of producing sugar-modified and/or alpha-phosphate modified nucleoside triphosphates, the method comprising: the PPK is incubated with the corresponding modified nucleoside monophosphates or nucleoside diphosphate and polyphosphate under suitable conditions.
  17. 17. The method of claim 16, further comprising incubating with divalent cations.
  18. 18. The method of claim 17, wherein the divalent cation is Mg 2+ 、Mn 2+ or Co 2+ .
  19. 19. The method of claim 16, further comprising incubating with magnesium chloride.
  20. 20. The method of any one of claims 16 to 19, wherein the PPK is selected using the method of any one of claims 6 to 15.

Description

Method for producing Nucleoside Triphosphates (NTPs) Technical Field The present invention relates to novel enzymatic methods for producing Nucleoside Triphosphates (NTPs), in particular modified NTPs. NTPs produced by the methods of the invention may be used in methods of producing polynucleotides (including oligonucleotides) for treatment. Background Nucleoside Triphosphates (NTPs) are fundamental building blocks of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Naturally occurring NTPs include Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), thymidine Triphosphate (TTP), and Uridine Triphosphate (UTP). ATP is a major source of cellular energy, and GTP is often used as a cofactor for enzymes and proteins. Commercial supplies of naturally occurring NTPs are typically obtained using cell fermentation and purification methods. NTPs with modifications to sugar, base, and/or phosphate groups are valuable laboratory tools and are also therapeutically useful, for example, in the preparation of nucleotide-based therapeutics (e.g., therapeutic oligonucleotides and mRNA) because they help to enhance affinity for targets, improve nuclease resistance, alter circulation characteristics, and modulate immune characteristics (Kulkarni et al Nature Nanotechnology, 2021, 16, 630-643). Some wild-type polymerases as well as engineered polymerases are capable of utilizing modified NTPs to generate modified polynucleotides complementary to a template sequence (Milisavljevic et al., org. Biomol. Chem, 2018, 16, 5800-5807; and Sabat et al., front Chem., 2023, 11, 1161462). In addition, terminal deoxynucleotidyl transferase (TdT) can utilize modified NTPs and produce modified oligonucleotides in a template independent manner (WO 2019/121500). However, modified NTPs are difficult to produce and expensive to produce, especially for large-scale production. Synthetic reaction schemes for producing modified NTPs are listed in the 2012 reviews paper (Molecules, 2012, 17, 13569-13591) of Marcel Hollenstein and include the Yoshikawa scheme, the Ludwig-Eckstein method, the Borch method, and other strategies (see also Burgess and Cook, chem. Rev 2000, 100, 2047-2059). Recently, biocatalytic methods for the production of specific NTPs have been investigated. MK-1454 is a cyclic dinucleotide Stimulator (STING) agonist of the interferon gene, which is being developed as an immune tumor therapeutic. Starting from two unnatural nucleoside phosphorothioates, MK-1454 has been diastereoselectively assembled by a one-pot cascade in which two phosphorothioate nucleotides are produced simultaneously by biocatalysis followed by coupling and cyclization by catalysis with engineered animal cyclic guanosine-adenosine synthases (cGAS). For phosphorothioate synthesis, three kinases were engineered to develop an unnatural cofactor recycling system (McIntosh et al, nature, 2022, 603, 439-444). However, there is a need for simpler and more cost-effective methods to generate NTPs, particularly modified NTPs, which can be used on a large scale, particularly in the preparation of polynucleotide therapeutics. Summary of The Invention In a first aspect of the invention there is provided the use of a polyphosphate kinase (PPK) in the production of a sugar-modified and/or alpha-phosphate modified nucleoside triphosphate, wherein the sugar-modified and/or alpha-phosphate modified nucleoside triphosphate is produced from a corresponding modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate. In a second aspect of the invention, a method is provided for screening a set of putative and known PPKs to identify enzymes capable of producing a correspondingly modified nucleoside triphosphate using a sugar-modified and/or alpha-phosphate modified nucleoside monophosphate or nucleoside diphosphate as a substrate. The method comprises a) identifying sequences encoding the set of putative and known PPKs, b) synthesizing the PPKs, c) purifying the PPKs, d) mixing each of the purified PPKs with the sugar-modified and/or alpha-phosphate-modified nucleoside mono-or di-phosphates and polyphosphates to form reaction mixtures, and e) analyzing each reaction mixture for the presence of a corresponding modified nucleoside triphosphate to determine which enzyme or enzymes produce the corresponding modified nucleoside triphosphates. In a third aspect of the invention, there is provided an enzymatic method of producing sugar-modified and/or alpha-phosphate modified nucleoside triphosphates, the method comprising incubating PPK with the corresponding modified nucleoside monophosphates or nucleoside diphosphate and polyphosphate under suitable conditions. In a fourth aspect of the invention there is provided the use of a modified nucleoside triphosphate obtained by the method of the invention in the preparation of a polynucleotide. In a fifth aspect of the invention there is provided the use of PPK to produce stereospecif