WO2025242702A1 - Procédé enzymatique de production de groupes de n-acétyl galactosamine - Google Patents

Procédé enzymatique de production de groupes de n-acétyl galactosamine

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WO2025242702A1
WO2025242702A1 PCT/EP2025/063914 EP2025063914W WO2025242702A1 WO 2025242702 A1 WO2025242702 A1 WO 2025242702A1 EP 2025063914 W EP2025063914 W EP 2025063914W WO 2025242702 A1 WO2025242702 A1 WO 2025242702A1
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nitrilase
galnac
oligonucleotide
seq
cluster
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Douglas E. FUERST
Christopher Matthew MACDERMAID
James Patrick MORRISON
Anthony Joseph NOCKET
Gheorghe Doru ROIBAN
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GlaxoSmithKline Intellectual Property Development Ltd
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GlaxoSmithKline Intellectual Property Development Ltd
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • ENZYMATIC PROCESS FIELD OF THE INVENTION T he invention relates to a novel process for generating N-acetylgalactosamine (GalNAc) clusters using a nitrilase and conjugating said GalNAc clusters to oligonucleotides, such as oligonucleotides for use in therapy.
  • GalNAc N-acetylgalactosamine
  • BACKGROUND Oligonucleotides have emerged as an important new class of therapeutic drugs which have the ability to treat a wide range of diseases.
  • siRNAs can silence mRNA via an antisense mechanism, triggering extraordinar selectivity for genes of interest and blocking the transfer of genetic information from DNA to protein, or recruit the RNA-induced silencing complex and downregulate protein expression, e.g., siRNAs (Stein and Castanotto, 2017, Mol. Ther.25(5), 1069-1075; Mullard, 2018, Nat. Rev. Drug Discov.17(9), 613; Roberts et al, 2020, Nat. Rev. Drug Discov.19, 673-694).
  • siRNAs Stein and Castanotto, 2017, Mol. Ther.25(5), 1069-1075; Mullard, 2018, Nat. Rev. Drug Discov.17(9), 613; Roberts et al, 2020, Nat. Rev. Drug Discov.19, 673-694.
  • U nlike small molecular agents which are lipophilic and readily taken up by tissues and cells, the majority of oligonucleotide therapeutics are larger in size, more hydrophilic and their cellular uptake remains
  • the liver is the largest internal organ in the human body, playing vital functions in metabolism, detoxification, and iron homeostasis, as well as synthesis and secretion of major plasma proteins, with over 80% of its mass composed of hepatocytes. It is therefore not surprising that large amounts of disease targets reside within the liver hepatocytes (Cui et al, 2021, ACS Omega, 6, 16259-16265).
  • GalNAc N -acetylgalactosamine binds to asialoglycoprotein receptors (ASGPRs) (KD ⁇ 2.5 nM) on hepatocytes and conjugation of oligonucleotides to clusters of GalNAc units more effectively targets the drug to the liver and enables hepatocyte uptake via ASGP receptor-mediated endocytosis (Huang, 2017, Mol. Ther. Nucl. Acids 6, 116-132; Prakash et al, 2014, 42(13), 8796-8807). This can lead to lower doses, thereby increasing the therapeutic index and improving the affordability of the medicine by reducing cost of goods (Wang et al, 2019, Excerpt Opin.
  • GalNAc oligonucleotide conjugate was the first GalNAc oligonucleotide conjugate to be registered and was approved for use in the treatment of acute hepatic porphyria (AHP) in the US in 2019 and in Europe in 2020.
  • AHP acute hepatic porphyria
  • GalNAc clusters are designed to give the appropriate spatial arrangement for optimal binding to the hepatocyte receptor, with the optimal presentation distance between each GalNAc thought to be 15–20 ⁇ (Debacker et al, 2020, Mol. Ther.
  • GalNAc clusters may be divalent, trivalent or tetravalent, although trivalent GalNAcs are the most commonly used. G alNAc clusters have a high degree of entropy and are therefore not typically crystalline. Crystallisation is often exploited by synthetic chemists to reduce the levels of impurities but, for GalNAc clusters, alternative strategies are required to ensure quality, especially if it is desirable to avoid chromatographic purification. Current methods for making GalNAc clusters are multi-step chemical reaction schemes with various difficult to separate by-products (Prakash et al, 2016, J. Med. Chem., 59(6), 2718-2733; Kim et al, 2023, Org, Process. Res.
  • the present invention provides use of a nitrilase in the manufacture of an N-acetylgalactosamine (GalNAc) cluster.
  • the present invention provides use of a nitrilase to form a compound of formula (I): from a compound of formula (II): , wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide.
  • the present invention provides a method of producing a compound of formula (I): by reacting a compound of formula (II): with a nitrilase under suitable conditions, wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide.
  • the present invention provides a method of producing a compound of formula (III): by reacting a compound of formula (IV): with a nitrilase under suitable conditions, wherein L is a leaving group such as C1-20alkyl, C1- 20alkoxy or benzyl.
  • the present invention provides a method of manufacturing a GalNAc-conjugated oligonucleotide comprising conjugating a GalNAc cluster to said oligonucleotide under suitable conditions, wherein said GalNAc cluster is made using a nitrilase.
  • the present invention provides a GalNAc-conjugated oligonucleotide for use in therapy, wherein the GalNAc-conjugated oligonucleotide comprises a GalNAc cluster made using a nitrilase.
  • the present invention provides a method of producing a tris-based GalNAc cluster compound of formula (V): wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide and A is a group comprising GalNAc, the method comprising reacting a compound of formula (II): with a nitrilase under suitable conditions to obtain a triacid intermediate, then reacting the triacid intermediate with a compound A-L2 wherein L2 is a leaving group.
  • the invention provides a GalNAc-conjugated therapeutic oligonucleotide obtained as described herein.
  • the present invention provides an engineered nitrilase comprising a sequence that is at least 90% identical to SEQ ID NO:1 and includes a catalytic triad consisting of E, K and C at positions equivalent to E57, K147 and C181 in SEQ ID NO:1.
  • the present invention provides an engineered nitrilase comprising or consisting of an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.
  • the present invention provides an engineered nitrilase comprising a sequence that is at least 90% identical to SEQ ID NO:1 and includes a catalytic triad consisting of Glu, Lys, and Cys, wherein the engineered nitrilase exhibits improved nitrilase activity relative to the nitrilase of SEQ ID NO:1 for converting a trinitrile substrate to a triacid product.
  • the present invention provides a polynucleotide encoding the engineered nitrilase according to the present invention.
  • the present invention provides an expression vector comprising the polynucleotide according to the present invention.
  • the present invention provides a host cell comprising the expression vector according to the present invention.
  • the present invention provides a method of preparing an engineered nitrilase comprising culturing a host cell according to the present invention under suitable conditions.
  • the present invention provides a method of screening a panel of nitrilases to identify an enzyme capable of catalysing the conversion of a compound of wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide; comprising: a . identifying sequences encoding said panel of nitrilases; b. synthesizing said nitrilases; c.
  • DESCRIPTION OF DRAWINGS/FIGURES F IG.1 is a general reaction scheme for the nitrilase catalysed hydrolysis of organic nitriles (-C ⁇ N) into carboxylic acids and free amine (and amides under certain conditions).
  • F IG.2 is reaction scheme for generating an exemplary GalNAc cluster depicting chemical versus enzymatic routes (Scheme 1).
  • FIG.3 is a reaction scheme for the nitrilase hydrolysis of trinitriles 3 and 4 and the identified products (Scheme 2).
  • FIG.4 (A) shows the amino acid distribution within the nitrilase active site from hits NIT1-D12, NIT1-F11, NIT1-G2, and NIT1-H8, 1524 homologues, the selected NIT2 panel and NIT2-D1.
  • B shows the location of W158 in the active site of NIT1-D12 homology model on 3WUY.
  • Catalytic residues (E57, K147, C181) are shown as spheres and residues lining the active site are shown as sticks. Position 158, is predominantly tryptophan in identified homologues. Mutation of this position away from tryptophan has been observed to drive formation of the amide side product.
  • F IG. 5 shows the largest fold improvement (FI) found for each position in the nitrilase after one round of directed evolution (Rd01) with and fatter tubes indicating larger FIs.
  • GalNAc as used herein is short for N-acetylgalactosamine, also known as 2- (acetylamino)-2-deoxy-D-galactose (IUPAC), the structure of which is shown below.
  • IUPAC 2- (acetylamino)-2-deoxy-D-galactose
  • the three hydroxy groups in the N-acetylgalactosamine moiety may be independently acetylated.
  • the N-acetylgalactosamine group may be: (where none of the hydroxy groups are acetylated), or (when all three hydroxy groups are acetylated), or any other combination of acetylation.
  • the GalNAc cluster is trivalent.
  • GalNAc cluster as used herein comprises a chemical scaffold with two or more GalNAc molecules attached thereto.
  • a GalNAc cluster is a multivalent ligand for an asialoglycoprotein receptor (ASGPR).
  • GalNAc clusters may be divalent (two GalNAc molecules attached to the scaffold), trivalent (three GalNAc molecules attached to the scaffold, also known as triantennary GalNAc) or tetravalent (four GalNAc molecules attached to the scaffold).
  • the affinity of the ASGPR for a trimer of GalNAc is 1,000-fold higher than a dimer, and 1,000-fold higher than a monomer, while a tetramer has just a slightly higher affinity for the receptor than a trimer.
  • GalNAc cluster is a GalNAc cluster derived from tris buffer (2-amino-2- (hydroxymethyl)-1,3-propanediol).
  • Nitrilase as used herein is an enzyme, also known as a nitrile aminohydrolase, that catalyses the hydrolysis of organic nitriles (-C ⁇ N) into carboxylic acids and free amine (and amides under certain conditions): RCN + 2H 2 O ⁇ RCO 2 H + NH 3 (see also Figure 1). Nitrilases are covered in EC 3.5.5.1.
  • a nitrilase is usually a single polypeptide ranging from 32-45 kDa with an ⁇ - ⁇ - ⁇ - ⁇ fold structure. The most favoured form is a filament consisting of 6-26 subunits.
  • Nitrilase enzymes belong to the thiol hydrolases class possessing the Lys (K)- Cys (C)-Glu (E) catalytic triad which is essential for its active site function.
  • Oligo for short, as used herein means a polymer of nucleotide residues.
  • oligonucleotide is usually used for shorter sequences than the term “polynucleotide”, generally in the range of 3 to 30 nucleotides.
  • Oligos can be single-stranded, such as antisense molecules, or double-stranded, such as siRNA molecules.
  • An oligonucleotide may be conjugated to another molecule, e.g., N- Acetylgalactosamine (GalNAc) or multiples thereof (e.g., a GalNAc cluster).
  • GalNAc N- Acetylgalactosamine
  • Percent identity or “% identity” between a query amino acid sequence and a subject amino acid sequence is the “Identities” value, expressed as a percentage, that is calculated using a suitable algorithm (e.g. BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR) or software (e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g. Needleman-Wunsch or GenePAST/KERR) or software (e.g. DNASTAR Lasergene or GenePAST/KERR).
  • a suitable algorithm e.g. BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR
  • software e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign
  • a query amino acid sequence may be described by an amino acid sequence disclosed herein, in particular in one or more of the claims, for example SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
  • the query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid alterations as compared to the subject sequence such that the % identity is less than 100%.
  • the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence.
  • Such alterations include at least one amino acid residue deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein said alterations may occur at the amino- or carboxy- terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acid residues in the query sequence or in one or more contiguous groups within the query sequence.
  • “Scaffold” as used herein is the core chemical structure within a GalNAc cluster. Examples of scaffolds include those derived from tris buffer (2-amino-2-(hydroxymethyl)-1,3- propanediol).
  • “Therapeutic oligonucleotide” or “therapeutic oligo” as used herein means an oligonucleotide that has a therapeutic application, e.g., in the prevention or treatment of a condition or disease in a human or animal. Therapeutic oligos typically contains one or more modified nucleotide residues or linkages.
  • Therapeutic oligos act via one of several different mechanisms, including, but not limited to, antisense, splice-switching or exon-skipping, immunostimulation, RNA interference (RNAi), e.g., via microRNA (miRNA) or small interfering RNA (siRNA), and recruitment and guiding of DNA and RNA editing enzymes, for example an A to I RNA base-editing oligonucleotide (AIMer).
  • a therapeutic oligo may be an aptamer.
  • Therapeutic oligonucleotides will usually, but not always, have a defined sequence.
  • substitution refers to a change in the amino acid residue at a specified position of a polypeptide sequence when compared to a reference sequence, such as the wild-type sequence or another engineered variant.
  • Constant amino acid substitutions or mutations refer to the interchangeability of residues having similar side chains, and thus typically involves the substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids.
  • conservative mutations do not include substitutions from a hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl- containing to hydroxyl-containing, or small to small residue, if the conservative mutation can instead be a substitution from an aliphatic to an aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or constrained to constrained residue.
  • A, V, L, or I can be conservatively mutated to either another aliphatic residue or to another non-polar residue. Table 1 below shows exemplary conservative substitutions.
  • N itrilases are useful in the manufacture of N-acetylgalactosamine (GalNAc) clusters.
  • the inventors have designed a convergent process for the synthesis of GalNAc clusters.
  • the GalNAc cluster produced in this process are crystalline, which aids the control of impurities.
  • N itrilases are enzymes that catalyse the hydrolysis of organic nitriles (-C ⁇ N) into carboxylic acids and free amine, and amides under certain conditions (see Figure 1).
  • the electrophilic carbon of the nitrile is subject to nucleophilic attack by one of the two SH groups on the nitrilase (EnzSH).
  • the thioimidate formed is subsequently hydrolyzed to the acylenzyme (SEnz) and ammonia is created as a by-product.
  • the acylenzyme can undergo one of two pathways depending on the conditions: (i) further hydrolyzation of the acylenzyme with water produces the carboxylic acid and the regenerated enzyme, or (ii) the acylenzyme is hydrolyzed by ammonia, displacing the enzyme and forming the amide product.
  • Conditions that promote amide formation include early release of the enzyme-bound substrate after the first water hydrolysis followed by delayed addition of the second water molecule, low temperature and increased pH conditions (for bioconversions by nitrilase for most bacteria and fungi, the optimal pH range is between 7.0-8.0 and the optimal temperature range is between 30-50°C) and electron withdrawing groups at the ⁇ -position.
  • Nitrilases have previously been successfully used to manufacture targets ranging from commodity chemicals, such as acrylic acid, to intermediates in the synthesis of atorvastatin (LIPITOR) and pregabalin (LYRICA). However, despite their well-established utility in the synthesis of simple intermediates, implementation of nitrilases in the preparation of bulky substrates has been limited.
  • T he published method of chemically synthesising a tris based GalNAc cluster involves a Michael addition of THAM (tris(hydroxymethyl)aminomethane) with tert-butyl acrylate.
  • THAM tris(hydroxymethyl)aminomethane
  • intermediate triacid 6 (3,3'-((2-(5-(Benzyloxy)-5-oxopentanamido)-2-((2-carboxyethoxy)methyl)propane-1,3- diyl)bis(oxy))dipropionic acid) is an effective control point.
  • W hen generating the triacid from the trinitrile it is desirable to use mild conditions to prevent side reactions, but selective nitrile hydrolysis is difficult to achieve. Strong acids are well known to carry out nitrile hydrolysis, but these reaction conditions are incompatible with the other functional groups in the trinitrile and can generate undesirable by-products, e.g., via concomitant ester hydrolysis.
  • a nitrilase to form a compound of formula (I): from a compound of formula (II): , wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide.
  • a linker group suitable for attaching to a therapeutic oligonucleotide will inevitably also be suitable for attaching to a protected version of the therapeutic oligonucleotide, or to a portion of the therapeutic oligonucleotide, for example, an oligonucleotide monomer (e.g. a phosphoroamidite) that will be incorporated into the therapeutic oligonucleotide via solid phase synthesis, or a “shortmer” (a portion of the oligonucleotide, which forms the therapeutic oligonucleotide once ligated to one or more other shortmers), or to protected versions thereof.
  • an oligonucleotide monomer e.g. a phosphoroamidite
  • a shortmer a portion of the oligonucleotide, which forms the therapeutic oligonucleotide once ligated to one or more other shortmers
  • the linker group R (attached to the GalNAc cluster) enables the ultimate attachment of the GalNAc cluster to therapeutic oligonucleotides, for hepatic delivery of the attached oligonucleotide via the asialoglycyprotein receptor on hepatocytes.
  • nitrile i.e. the compounds of formulae II, IV or VII
  • acid i.e. the compound of formulae I, III or VI
  • the precursor for example on the oligonucleotide monomer, shortmer or therapeutic oligonucleotide.
  • R may be any moiety suitable for attaching the GalNAc cluster to a therapeutic oligonucleotide, by acting as a linker between the GalNAc cluster and the oligonucleotide or a portion thereof.
  • R as used herein may comprise part of, or all of, the linker between the GalNAc cluster and the oligonucleotide, depending on the final structure of the conjugated oligonucleotide.
  • R may end in a hydrogen or any suitable leaving group such as alkyl or aryl, e.g. C 1-20 alkyl or benzyl.
  • the compound of formula (I) according to the present invention is selected from: , wherein L is a leaving group, such as C1-20alkyl, C1-20alkoxy or benzyl. In one embodiment L is C 1-6 alkyl or benzyl.
  • the reaction temperature of the nitrilase as described herein is from 30 oC to 45 oC. In an embodiment, the reaction temperature is from 30 oC to 40 oC. In an embodiment, the reaction temperature is about 30 oC.
  • the reaction pH is between 4 and 10, between 5 and 9, or between 6 and 8. In an embodiment, the reaction pH is about 7.
  • the triacid of formula (I) is crystallisable.
  • the compound of formula (II) is: , wherein L 1 is ethyl or benzyl, and the compound of formula (I) is: .
  • the compound of formula (II) is: and the compound of formula (I) is: .
  • the compound of formula (II) is: and the compound of formula (I) is:
  • the compound of formula (II) is: and the compound of formula (I) is: .
  • the triacid compound of formula (I) is formed as described herein, it may be reacted with any appropriate moiety comprising GalNAc using standard chemistry known to the skilled person to form a GalNAc cluster suitable for ultimately conjugating to a therapeutic oligonucleotide or a portion thereof.
  • the compound A-L 2 is: wherein L2 is a leaving group such as benzyl and each of R1, R2 and R3 is independently hydrogen or acyl.
  • R1, R2 and R3 are each hydrogen.
  • the compound A-L2 is: wherein L2 is a leaving group such as benzyl and each of R1, R2 and R3 is independently hydrogen or acyl. In one embodiment, R1, R2 and R3 are each hydrogen.
  • the trivalent GalNAc cluster formed from the triacid compound of formula (I) is: wherein G is N-acetylgalactosamine, n is 0 to 10, and R is a linker group suitable for attaching to a therapeutic oligonucleotide.
  • the compound of formula (V) is selected from: wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide. In an embodiment, the compound of formula (V) is selected from: ,
  • the nitrilase is NIT2-D1 (SEQ ID NO:1).
  • the nitrilase is M1 (SEQ ID NO:2).
  • the nitrilase is M2 (SEQ ID NO:3).
  • the nitrilase comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
  • the nitrilase comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, wherein the difference is a conservative substitution of the amino acid(s).
  • the nitrilase comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:1.
  • the nitrilase comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:2.
  • the nitrilase comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:3.
  • the nitrilase is an engineered nitrilase.
  • the nitrilase is present at ⁇ 10% w/w and complete conversion of substrate to products is obtained in ⁇ 24 hours with ⁇ 0.20% a/a impurities.
  • Methods of engineering enzymes are known to persons skilled in the art and include mutagenesis and/or DNA shuffling, as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and U.S. Patent No. 6,537,746.
  • an engineered nitrilase comprising a sequence that is at least 90% identical to SEQ ID NO:1 and includes a catalytic triad consisting of E, K and C at positions equivalent to E57, K147 and C181 in SEQ ID NO:1.
  • the engineered nitrilase comprises a sequence that is at least 90% identical to SEQ ID NO:1 and includes a catalytic triad consisting of E, K and C at positions equivalent to E57, K147 and C181 in SEQ ID NO:1.
  • the amino acid at a position equivalent to position 158 in SEQ ID NO:1 is an aromatic amino acid.
  • the aromatic amino acid is selected from the group consisting of: F, H, W and Y.
  • any other amino acid substitutions are conservative substitutions.
  • one or more amino acids at positions equivalent to the following positions in SEQ ID NO:1 have been mutated: 9, 56, 58, 61, 94, 137, 138, 141, 183, 192, 242, 243, 258, 275, 278, 305.
  • 1, 2, 3 or 4 amino acids at any position equivalent to the following positions in SEQ ID NO:1 have been mutated: 9, 56, 58, 61, 94, 137, 138, 141, 183, 192, 242, 243, 258, 275, 278, 305.
  • amino acid position 9 is L or Y; position 56 is C, F or V; position 58 is K, N or W; position 59 is V; position 61 is E, H, I, L or M; position 94 is R; position 137 is A, C, D, E, F, G, L, M, S, T, W, or Y; position 138 is A, C, E, F, L, M, R, W or Y; position 141 is I; position 183 is C; position 192 is G; position 209 is A; position 242 is I; position 243 is S; position 258 is A; position 275 is N; position 278 is M; and/or position 205 is L; wherein the foregoing amino acid positions are numbered with reference to SEQ ID NO:1.
  • the engineered nitrilase comprises G at a position equivalent to position 192 in SEQ ID NO:1 and a set of mutations as set out in any row of Table 4.
  • an engineered nitrilase comprising or consisting of an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 is provided.
  • an engineered nitrilase comprising a sequence that is at least 90% identical to SEQ ID NO:1 and includes a catalytic triad consisting of Glu, Lys, and Cys, wherein the engineered nitrilase exhibits improved nitrilase activity relative to the nitrilase of SEQ ID NO:1 for converting a trinitrile substrate to a triacid product.
  • the improved nitrilase activity is at least 1.1 fold improved activity for converting a trinitrile substrate to a triacid product.
  • the engineered nitrilase has reduced side product formation relative to SEQ ID NO:1.
  • the side product level is ⁇ 0.2% a/a.
  • the improved activity is complete conversion of the trinitrile product to triacid substrate in less than 24 hours.
  • the engineered nitrilase has reduced side product formation relative to SEQ ID NO:1 and complete conversion of the trinitrile product to triacid substrate in less than 24 hours.
  • Nitrilases disclosed herein may be prepared by any of a number of conventional techniques. For example, nitrilases may be purified from cells that naturally express them or produced in recombinant expression systems.
  • a polynucleotide encoding an engineered nitrilase as disclosed herein is provided.
  • an expression vector comprising a polynucleotide encoding an engineered nitrilase as disclosed herein is provided.
  • a host cell comprising the aforementioned expression vector is provided.
  • a number of different expression systems and purification regimes can be used to generate the nitrilases of the disclosure.
  • host cells are transformed with a recombinant expression vector encoding the desired nitrilase.
  • the expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome depending on the expression system.
  • a wide range of host cells can be employed, including Prokaryotes (including Gram negative or Gram positive bacteria, for example Escherichia coli, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi (for example Aspergilus sp.), or higher Eukaryotes, including insect cells and cell lines of mammalian origin (for example, CHO, NS0, PER.C6, HEK293, and HeLa).
  • the vector is a pET vector.
  • the vector is pET24b(+).
  • a method of preparing an engineered nitrilase comprising culturing a host cell under suitable conditions.
  • the cells can be cultured under conditions that promote expression of the nitrilase using a variety of equipment such as shake flasks, spinner flasks, and bioreactors.
  • a crude cell lysate or lyophilised powder may be used in experiments to demonstrate nitrilase activity.
  • cells containing the nitrilase are harvested by centrifugation and the supernatant is discarded.
  • the pellets are then resuspended, e.g., in potassium phosphate buffer, and re-suspended cultures are filtered, e.g., through two layers of muslin, lysed, e.g., by two passes through a microfluidizer, and cooled, e.g., with ice, with cell debris being removed, e.g., by centrifugation.
  • the lysate supernatant is collected, pooled, and lyophilized.
  • the nitrilase polypeptide is recovered by conventional protein downstream procedures and may be used in purified form.
  • a method of manufacturing a GalNAc-conjugated oligonucleotide comprising a step of making a GalNAc cluster using a nitrilase.
  • the invention provides a method of manufacturing a GalNAc-conjugated oligonucleotide is provided, comprising conjugating a GalNAc cluster to said oligonucleotide under suitable conditions, wherein said GalNAc cluster is made using a nitrilase.
  • the GalNAc is directly conjugated to a therapeutic oligonucleotide.
  • the GalNAc is conjugated to a protected oligonucleotide that can be deprotected to provide a GalNAc conjugated therapeutic oligonucleotide.
  • a method of manufacturing a GalNAc-conjugated therapeutic oligonucleotide comprising: a . manufacturing a GalNAc cluster using a nitrilase; b. conjugating said GalNAc cluster to a step to an oligonucleotide or a portion thereof; and c . manufacture of the GalNAc-conjugated therapeutic oligonucleotide. Steps a. and b. may be conducted as described herein.
  • the oligonucleotide in step b. is a protected version of the therapeutic oligonucleotide and step c. comprises deprotection.
  • step b. comprises conjugation of the GalNAc cluster to an oligonucleotide monomer or a protected version thereof (e.g. a phosphoroamidite).
  • the oligonucleotide monomer or protected version can be used in the manufacture of the GalNAc-conjugated therapeutic oligonucleotide using techniques well known in the art.
  • step b. comprises conjugation of the GalNAc cluster to a shortmer or a protected version thereof.
  • a method of manufacturing a GalNAc-conjugated therapeutic oligonucleotide comprising: a . manufacturing the carboxylic acid precursor to the GalNAc cluster using a nitrilase; b. conjugating said carboxylic acid precursor to an oligonucleotide or a portion thereof; and c . manufacture of the GalNAc-conjugated therapeutic oligonucleotide. Steps a. and b. may be conducted as described herein.
  • step c. would comprise formation of the GalNAc cluster and deprotection to form the therapeutic oligonucleotide.
  • step b. comprises conjugation of the carboxylic acid precursor to an oligonucleotide monomer or a protected version thereof.
  • the oligonucleotide monomer or protected version thereof can be used in the manufacture of the GalNAc-conjugated therapeutic oligonucleotide using techniques well known in the art.
  • the GalNAc cluster is formed on the therapeutic oligonucleotide.
  • a method of manufacturing a GalNAc-conjugated therapeutic oligonucleotide comprising: a . conjugating the nitrile precursor to a GalNAc cluster onto an oligonucleotide or a portion thereof; b . manufacture of the GalNAc-conjugated therapeutic oligonucleotide in a method using a nitrilase. Step a.
  • Step b. comprises a step of using a nitrilase to prepare the carboxylic acid precursor to the GalNAc cluster from the nitrile precursor. This step may take place at any time in the manufacture of the GalNAc-conjugated therapeutic oligonucleotide.
  • step b would further comprise formation of the GalNAc cluster and deprotection to form the therapeutic oligonucleotide.
  • the nitrile precursor is conjugated to an oligonucleotide monomer or protected version thereof (e.g.
  • the step of formation of the carboxylic acid precursor may take place after the phosphoroamidite is used in oligonucleotide synthesis.
  • the subsequent step of formation of the GalNAc cluster does not need to take place immediately following the step of preparing the carboxylic acid precursor.
  • the nitrile precursor it would be possible for the nitrile precursor to be conjugated to a phosphoroamidite, which is then used in shortmer synthesis.
  • the carboxylic acid conversion could take place on the shortmer, with the shortmer being used in oligonucleotide synthesis, with the step of formation of the GalNAc cluster occurring only as the final step.
  • the step of formation of the carboxylic acid precursor may take place before or after the shortmer or protected version thereof is used in oligonucleotide synthesis.
  • the subsequent step of formation of the GalNAc cluster does not need to take place immediately following the step of preparing the carboxylic acid precursor.
  • the nitrile precursor it would be possible for the nitrile precursor to be conjugated to a shortmer, which is then used in oligonucleotide synthesis, with the step of formation of the GalNAc cluster occurring after oligonucleotide synthesis.
  • GalNAc clusters as disclosed herein may be conjugated to oligonucleotides by a number of different methods known in the art, including amide coupling, phosphoramidite coupling and click-chemistry.
  • amide coupling Nucleic Acids Res., 2014, 42, 8796-8807
  • Nair et al J. Am. Chem. Soc., 2014, 136, 16958- 16961
  • Prakash et al J. Med. Chem., 2016, 59, 2718-2733
  • Matsuda et al ACS Che Biol, 2015, 10, 1181-1187; Farzan et al, Bioconjug.
  • the invention provides a GalNAc-conjugated therapeutic oligonucleotide obtained as described herein.
  • the oligonucleotide is selected from the group consisting of: an antisense oligonucleotide, siRNA, miRNA, aptamer, CRISPR guide RNA, and an oligonucleotide used to recruit and guide DNA and RNA editing enzymes.
  • the oligonucleotide used to recruit and guide DNA and RNA editing enzymes is an A to I RNA base-editing oligonucleotide (AIMer).
  • the oligonucleotide is a therapeutic oligonucleotide.
  • a GalNAc-conjugated oligonucleotide for use in therapy wherein the GalNAc-conjugated oligonucleotide comprises a GalNAc cluster made using a nitrilase.
  • the oligonucleotide is an antisense oligonucleotide.
  • the oligonucleotide is an siRNA.
  • the oligonucleotide is an miRNA.
  • the oligo nucleotide is an aptamer.
  • the oligonucleotide is a CRISPR guide RNA.
  • the oligonucleotide is an oligonucleotide used to recruit and guide DNA and RNA editing enzymes. In an embodiment, the oligonucleotide is an A to I RNA base-editing oligonucleotide (AIMer).
  • AIMer A to I RNA base-editing oligonucleotide
  • the GalNAc-conjugated oligonucleotide is selected from the group consisting of: cimdelirsen, donidalorsen, eplontersen, givosiran, inclisiran, lumasiran, olezarsen, pelacarsen, sapablursen, vupanorsen, vutrisiran, AZD8233, H-020, IONIS-FB- LRx/RG6299, IONIS-FXI-LRx, RG-125, SR-059, STP-135G, VIR-2218, WVE-006 and AB-729.
  • the GalNAc-conjugated oligonucleotide is AZD8233. In an embodiment, the GalNAc-conjugated oligonucleotide is selected from the group consisting of: givosiran, inclisiran, lumasiran, vutrisiran, H-020, RG-125, SR-059, VIR-2218 and WVE-006.
  • the GalNAc-conjugated oligonucleotide is selected from the group consisting of: cimdelirsen, donidalorsen, eplontersen, olezarsen, pelacarsen, sapablursen, vupanorsen, IONIS-FB-LRx/RG6299, and IONIS-FXI-LRx.
  • the GalNAc-conjugated oligonucleotide is STP-135G.
  • the GalNAc-conjugated oligonucleotide is selected from the group consisting of: plozasiran, fazirsiran, olpasiran, zodasiran, GSK4532990, daplosiran and tomligisiran.
  • Numbered Embodiments The invention may be described by means of the following numbered embodiments: 1. Use of a nitrilase in the manufacture of an N-acetylgalactosamine (GalNAc) cluster. 2. The use according to embodiment 1, wherein the GalNAc cluster is a trivalent cluster. 3. The use according to embodiment 2, wherein the trivalent cluster is a tris-based trivalent cluster. 4.
  • a nitrilase to form a compound of formula (I): from a compound of formula (II): , wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide.
  • R is a linker group suitable for attaching to a therapeutic oligonucleotide.
  • L is a leaving group, such as C 1-20 alkyl, C 1-20 alkoxy or benzyl. In one embodiment L is C 1-6 alkyl or benzyl.
  • L is C 1-6 alkyl or benzyl.
  • L is a leaving group such as C 1-20 alkyl, C 1- 20 alkoxy or benzyl.
  • the reaction temperature is 30 oC to 40 oC.
  • the method according to embodiment 18, wherein the reaction pH is about 7. 20.
  • a method of manufacturing a GalNAc-conjugated oligonucleotide comprising conjugating a GalNAc cluster to said oligonucleotide under suitable conditions, wherein said GalNAc cluster is made using a nitrilase.
  • An engineered nitrilase comprising a sequence that is at least 90% identical to SEQ ID NO:1 and includes a catalytic triad consisting of E, K and C at positions equivalent to E57, K147 and C181 in SEQ ID NO:1.
  • the engineered nitrilase according to embodiment 26 wherein the amino acid at a position equivalent to position 158 in SEQ ID NO:1 is an aromatic amino acid.
  • the aromatic amino acid is selected from the group consisting of: F, H, W and Y. 29.
  • 31 The engineered nitrilase according to any one of embodiments 26 to 30, wherein the nitrilase comprises G at a position equivalent to position 192 in SEQ ID NO:1 and a set of mutations as set out in any row of Table 5.
  • An engineered nitrilase comprising or consisting of an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.
  • An engineered nitrilase comprising a sequence that is at least 90% identical to SEQ ID NO:1 and includes a catalytic triad consisting of Glu, Lys, and Cys, wherein the engineered nitrilase exhibits improved nitrilase activity relative to the nitrilase of SEQ ID NO:1 for converting a trinitrile substrate to a triacid product.
  • the trinitrile substrate is a compound of formula (II): is a compound of formula (I): wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide.
  • 36. A polynucleotide encoding the engineered nitrilase according to any one of embodiments 26 to 35.
  • An expression vector comprising the polynucleotide according to embodiment 36.
  • a host cell comprising the expression vector according to embodiment 37. 39.
  • a method of preparing an engineered nitrilase comprising culturing a host cell according to embodiment 38 under suitable conditions.
  • 40 A method of screening a panel of nitrilases to identify an enzyme capable of catalysing the conversion of a compound of formula (II): wherein R is a linker group suitable for attaching to a therapeutic oligonucleotide; comprising: a . identifying sequences encoding said panel of nitrilases; b. synthesizing said nitrilases; c. isolating said nitrilases; d. mixing each of said isolated nitrilases with a compound of formula (II) under suitable conditions, to form a reaction mixture; and e .
  • the LC-MS was equipped with a Waters CSH C18 (2.1 mm x 30 mm, 1.7 ⁇ m) column and 0.02% trifluoroacetic acid in water (mobile phase A) and 0.02% trifluoroacetic acid in ACN (mobile phase B) at flow rate of 1.3 mL/min at a column temperature of 45 °C.
  • the run time was 2 min with gradient Table 2 as below. Compound elution was monitored using Evaporative Light-Scattering Detector and Mass Ions.
  • UV was monitored (210 to 350 nm) and the MS was scanned (100 to 1250 m/z) in positive and negative mode.
  • the capillary voltage was 1.6 kV for positive mode and 0.8 kV for negative mode.
  • the cone voltage was 7 V for ESI+ and 15 V for ESI-.
  • UV was monitored (210 to 350 nm) and the MS was scanned (100 to 1250 m/z) in positive and negative mode.
  • the capillary voltage was 1.6 kV for positive and 0.8 kV for negative mode.
  • the cone voltage was 7 V for ESI+ and 15 V for ESI-.
  • Example 1 Screening nitrilases for activity in trinitrile to triacid transformation to produce 3,3'-((2-((2-Carboxyethoxy)methyl)-2-(5-ethoxy-5-oxopentanamido)propane- 1,3-diyl)bis(oxy))dipropionic acid
  • An in-house nitrilase collection, nitrilase panel version 1 (NIT1) was screened for the reaction of trinitrile 3 substrate (ethyl 5-((1,3-bis(2-cyanoethoxy)-2-((2- cyanoethoxy)methyl)propan-2-yl)amino)-5-oxopentanoate), which was easily prepared from commercially available materials, to triacid 5 (3,3'-((2-((2-Carboxyethoxy)methyl)-2-(5- ethoxy-5-oxopentanamido)propane-1,3-diyl)bis(oxy))dipropi
  • Figure 3 is a reaction scheme (Scheme 2) for the nitrilase-catalysed hydrolysis of trinitriles 3 and 4 and identified products.
  • the following general screening protocol was used.
  • T he NIT1 panel of nitrilases were cloned into and expressed in E. coli BL-21(DE3) and crude cell lysates were used to test nitrilase activity.
  • Potassium phosphate buffer 100 ⁇ L/well, 100 mM, pH 7.0
  • NIT1 containing 100 ⁇ L of crude clarified cell lysate per well.
  • Substrate 3 in DMSO (final concentration 1.4 mg/mL 3, 3.3% v/v DMSO) was then added to this plate.
  • the reaction plate was then sealed using aluminium/polypropylene laminate heat seal tape and incubated at 30 °C, 500 RPM in an Infors MultiTron shaker.
  • a t 17 h and 47 h, 20 ⁇ L/well aliquots were taken and diluted into 180 ⁇ L/well 1:1 ACN:H2O in a shallow-well polypropylene plate (Corning 96).
  • NIT1-D12 Diluted reaction aliquots were incubated at room temperature with shaking 900 RPM for 5 min, then clarified by centrifugation (4000 RPM, 10 min) and 100 ⁇ L/well of supernatant transferred to a new shallow-well polypropylene plate before analysis by LC-MS Method 1.
  • the top four enzymes identified after performing the general screening protocol for hydrolysis of the trinitrile 3 (NIT1-D12, NIT1-F11, NIT1-G2, and NIT1-H8) were prepared as lyophilised powder for further reaction analysis.
  • N IT1-D12 provided excellent selectivity for the formation of mono acid 8 (80%) and diacid 10 (15%) (see Scheme 2, Figure 3).
  • N IT1-H8 showed higher activity but delivered primary amides 12 (45%) and 14 (5%) in addition to the desired carboxylic acids 5 (45%) and 10 (5%) at pH 7.0 after 24 h (see Scheme 2, Figure 3).
  • Primary amides are common side-products of nitrilase reactions and they accumulate in reactions, as they are not substrates for further hydrolysis by the enzyme (Jiang et al, 2017, Catal. Sci Technol., 7 (5), 1122-1128; Sosedov and Stolz, 2015, Microbiol. Biot.99(6), 2623-2635).
  • amides 12 and 14 were therefore identified as a key factor in selecting an appropriate enzyme, as these impurities could be considered critical quality attributes as derivatives of these impurities would be likely to contaminate the API if they could not be removed by downstream purification. Since the primary amide by-products could potentially arise via the attack of acyl-enzyme intermediate by ammonia/ammonium generated during the reaction course, it was envisaged that controlling pH of the buffer system could bias the product distribution in favour of acids 5, 8 and 10. Accordingly, each of the four nitrilases were screened from pH 4.0 to pH 10.0 for the hydrolysis of trinitrile ethyl ester 3 to investigate activity and product distribution.
  • Trinitrile ethyl ester 3 (10 mg) was charged to wells of a deep-well plate (final concentration 50 mg/mL 3).100 ⁇ l/well of the appropriate buffer (either sodium citrate (200 mM, pH 4.0), sodium citrate (200 mM, pH 5.0), MES (200 mM, pH 6.0), potassium phosphate buffer (200 mM, pH 7.0), potassium phosphate buffer (200 mM, pH 8.0), CHES (200 mM, pH 9.0), or CHES (200 mM, pH 10.0)) were then charged to the plate, followed by 100 ⁇ L/well of a lyophilised enzyme powder solution in deionized water (20 mg/mL stock concentration) of the nitrilases NIT1-D12, -F11, -G2, and -H8, such that each enzyme/pH combination was sampled once.
  • the appropriate buffer either sodium citrate (200 mM, pH 4.0), sodium citrate (200 mM
  • reaction plate was sealed with aluminium/polypropylene laminate heat seal tape and incubated at 30 °C, 300 RPM in a Kuhner shaker.
  • 20 ⁇ L/well aliquots were diluted into 180 ⁇ L/well 1:1 ACN:H2O in a shallow-well polypropylene plate.
  • Diluted reaction aliquots were incubated at room temperature with shaking 800 RPM for 10 min, then clarified by centrifugation (4000 RPM, 10 min) and 100 ⁇ L/well of supernatant was used in analysis by LC-MS Method 1. Attenuated reactivities were observed at the extreme pHs for both for NIT1-D12 and NIT1-H8.
  • NIT1-F11 and NIT-G2 Similar pH trends were observed for NIT1-F11 and NIT-G2 while conversion was lower. No significant decrease in amides 12 and 14 was observed for NIT1-H8.
  • NIT1-D12 was screened at different temperatures ranging from 30 °C to 45 °C.
  • Trinitrile ethyl ester 3 (10 mg) was charged to 8 x 1.5 mL HPLC vials, and 500 ⁇ L of nitrilase NIT1-D12 in potassium phosphate buffer (200 mM, pH 7.0) was added (final concentration 20 mg/mL 3, either 10 mg/mL or 25 mg/mL nitrilase NIT1-D12). Vials were incubated at 30 °C, 35 °C, 40 °C, or 45 °C, with shaking at 500 RPM in an Eppendorf thermomixer.
  • Example 2 ‘Fingerprinting’ nitrilase computational hit expansion strategy
  • Directed evolution can be inefficient and time consuming, especially when the starting and desired end points are sufficiently different that multiple rounds of evolution are required. Smarter ways of identifying better starting points for evolution than simply utilising the output from Example 1 were needed. Having successfully identified a collection of nitrilases that catalyse the desired transformation, the inventors recognised this as a unique opportunity to use these results to search for improved wild-type nitrilases with the aim of reducing the number of rounds of evolution necessary to generate a fit for purpose variant. A computational hit expansion strategy called ‘fingerprinting’ was employed.
  • PLIF protein-ligand interaction fingerprinting
  • PLIFs are generally used to identify similar ligands that bind to the same protein target, they can also be used to identify similar binding pockets or residue motifs across protein targets that will bind the same ligand.
  • a PLIF at its most basic is a compressed representation of three dimensional protein-ligand interactions and often takes the form of a one-dimensional list of positions in a protein that interact with a bound ligand. Each interaction is further annotated with chemical information to help classify it and can include atom types, charges, hydrogen bonds, contacting protein residue types, as well as other well-known molecular descriptors.
  • T o identify nitrilases that catalyze the hydrolysis of trinitriles 3 and 4 (benzyl 5-((1,3- bis(2-cyanoethoxy)-2-((2-cyanoethoxy)methyl)propan-2-yl)amino)-5-oxopentanoate), enzymes that have similar PLIFs to NIT1-D12 when bound to the substrate were investigated. Unfortunately, there were no available crystal structures with bound ligands that have similar molecular structures to compounds 3 and 4.
  • 3WUY was selected as the homology template since it has a medium to high-quality alignment matching 306 of 327 positions with minimal insertions and deletions which were regulated to regions of loop structure.3WUY was resolved at 3.1 ⁇ resolution, contains two copies of the nitrilase in the asymmetric unit, and has an identity to NIT1-D12 of 36%.
  • the nitrilase model’s active site does not contain a ligand, thus Chemical Computing Group’s SiteFinder was used to identify the active site via cavity detection which was subsequently corroborated by the presence of the well-known catalytic triad of E57, K147 and C181.
  • the homology model was used to identify positions lining the nitrilase active site by selecting any residues having atoms within 6.0 ⁇ of the catalytic residues E57, K147 and C181. Twenty-two such positions were identified: 56, 57, 58, 63, 130, 146, 147, 148, 149, 150, 151, 154, 158, 180, 181, 182, 183, 204, 205, 206, 207 and 232.
  • a comparison of the top four NIT1 enzymes revealed identities between NIT1-D12 and -F11 of 78%, and -H8 and -G2 of 55% and 30%, respectively.
  • NIT1-D12, -F11 and -G2 The alignment of the NIT1 enzymes producing the desired product (NIT1-D12, -F11 and -G2) and the most active enzyme (NIT1-H8) and the homology template, showed -D12 and -F11 as having identical active sites while -H8 contains 3 mutations with respect to -D12: A58S, V232T and W158H.
  • the catalytic residues E57, K147, and C181 are all conserved across the five enzymes of interest (including 3WUY), as well as positions Y63, L148, P150, T151, E154, and E183.
  • the diversity of the active site fingerprinting was expanded to include the amino acid types at the 22 active site positions from not only the NIT1 hits, but the homologues and the homology model as well.
  • a two-step hierarchical clustering leveraging this expanded set of diversity was then performed.
  • the clustering was limited to only the fingerprint positions with a minimum cluster of 80% identity, essentially allowing active sites with up to 4 mutations to be clustered together, as an attempt to isolate the various active site motifs. This resulted in the initial hits being segmented into two clusters, one with NIT1-D12, -F11 and -H8 along with 192 other enzymes, and another with NIT1-G2 and 39 additional enzymes, for a total of 231.
  • a second, full-length sequence clustering step was then performed on the isolated 231 nitrilases at 70% identity, from which 48 disparate exemplars were selected from each cluster, enabling enzymes to be successfully identified that retain a high sequence identity in the active site, evenly sample aromatic diversity at position 158, in addition to varying levels of diversity at 257 other positions across the enzyme.
  • the fingerprint approach resulted in identification of 48 homologous wild-type enzymes active in the trinitrile to triacid transformation (nitrilase panel version 2, NIT2).
  • NIT2 trinitrile to triacid transformation
  • none of the 48 selected enzymes has previously been characterized in the literature to perform nitrile hydrolysis.
  • the active site diversity sampled from the 48 identified homologues with respect to NIT1-D12 is shown in Table 4.
  • Codon-optimized homologue genes for the new nitrilase collection were synthesized and cloned into pET24b(+). Plasmids were transformed into BL-21(DE3) Gold cells (Agilent) for expression and used for homologue screening against benzyl ester 4, which was selected over the ethyl ester 3 to facilitate a more facile deprotection step downstream in the synthesis. S ix enzymes that performed better than NIT1-D12 were identified. Further screening helped select the wild-type nitrilase NIT2-D1 (SEQ ID NO:1) from Acidithiobacillales bacterium SG8_45 (GenBank: KPK12037.1) as the best candidate.
  • Figure 4 (A) shows the amino acid distribution within the nitrilase active site from hits NIT1-D12, -F11 -G2, and -H8, 1524 homologues, the selected NIT2 panel and NIT2-D1.
  • Figure 4(B) shows the location of W158 in the active site of NIT1-D12 homology model on 3WUY. Catalytic residues E57, K147, C181 are shown by yellow spheres and residues lining the active site are cyan sticks. Position 158, highlighted in green, is predominantly tryptophan in identified homologues. Mutation of this position away from tryptophan has been observed to drive formation of the amide side product.
  • Example 3 Directed evolution of nitrilase for the production of 3,3'-((2-(5-(benzyloxy)- 5-oxopentanamido)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid
  • NIT2-D1, SEQ ID NO:1 identified using the fingerprint approach as set out in Example 2, having improved activity and selectivity over the initial hits from Example 1, was further improved using directed evolution.
  • Site saturation was chosen as the evolution strategy with the goal of identifying beneficial diversity that could be recombined later. This focused approach was preferred to other approaches such as untargeted and codon biased error prone PCR methodology to ensure quick success.
  • NIT2-D1 codon optimized for expression in E. coli was designed based on the reported amino acid sequence of the Acidithiobacillales bacterium SG8_45 homologue (GenBank: KPK12037.1). The gene was cloned into the expression vector pCK110900 under control of a lac promoter. Round 1 of evolution (Rd01) The main objective of the first round of evolution was to identify nitrilase variants with increased activity for the conversion of trinitrile 4 to triacid 6 which also reduce the formation of impurity 13.
  • FI fold improvement defined as variant activity divided by the average activity of positive controls in the same plate.
  • a library comprising mutations lining the active site delivered the highest FI for triacid 6 formation (up to 26.7 for variant N137C), with a few other variants following closely (D192G, N243S or R209A).
  • Position N137 was identified as a hotspot with N137F and N137T both showing excellent FI.
  • the second active shell gave excellent hits such as R56C, P57C and R56F, while other second shell positions also provided promising variants - with W61 being a clear hotspot.
  • Variant D192G (M1, SEQ ID NO:2) was selected as the Rd02 backbone as it gave the highest conversion of trinitrile 4 to triacid 6 at 2% & 10% w/w biocatalyst loading after 20 h on 0.5 g (of 4) scale. It also gave the second lowest amount of impurity 13 at full conversion. Following process development 10% w/w loading of M1 was used on 47.5 g (of 4) scale and 50 g/L 4, affording full conversion after 21 h with 71.4% yield and 0.23% a/a amide 13. Despite the improved properties of M1 over the initial NIT2-D1, another round was required to meet the process criteria.
  • the inventors developed protein-based pharmacophores also known as “fingerprints” which allowed them to generate new nitrilase variants with a minimally perturbed active site and diversity throughout the remainder of the enzyme.
  • a fter only two rounds of directed evolution a new fit for purpose enzyme was developed, which showed 25-fold improvement in activity under process conditions as when compared to the parent and importantly also possessed improved selectivity for triacid synthesis over primary amide critical impurity formation. Accordingly, the inventors have shown that the combination of active site fingerprinting with focused directed evolution is a technique that quickly delivers fit for purpose variants.
  • T he engineered nitrilase enzyme has enabled a new scalable route to the GalNAc cluster.
  • Nitrilase M2 is able to produce high-purity (>99.8%) triacid 6, a key crystallizable intermediate in the manufacture of the GalNAc cluster, from trinitrile 4. Further experiments E ngineered enzyme M2 was subsequently utilised in Examples 4, 5 and 6 to demonstrate its wider utility in accepting other bulky trinitriles and converting them to their corresponding crystallizable triacids, which in turn can be used in the production of further trivalent GalNAc clusters. The suitability of the combination of active site fingerprinting with focused directed evolution to further increase the activity and selectivity of the nitrilase for a given trinitrile has been demonstrated herein.
  • Example 4 Preparation of 3,3'-((2-(12-(benzyloxy)-12-oxododecanamido)-2-((2- carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid using a nitrilase N itrilase M2 (220 mg) was weighed into a Falcon tube and resuspended in potassium phosphate buffer (10 mL, 100 mM, pH 7.0) to a concentration of 22 mg/mL.900 ⁇ L of this solution was transferred to an Eppendorf Tube. Toluene (100 ⁇ L) was then added to the reaction mixture.
  • the reaction was started by the addition of benzyl 12-((1,3-bis(2-cyanoethoxy)-2-((2- cyanoethoxy)methyl)propan-2-yl)amino)-12-oxododecanoate trinitrile (4 ⁇ L) and incubating at 30 °C with shaking at 900 RPM in an Eppendorf Thermomixer. After 16 hours an aliquot (100 ⁇ L) was taken and diluted into 900 ⁇ L 1:1 ACN:H 2 O in an Eppendorf tube. The sample was clarified by centrifugation (10000 RPM for 1 minute) and the supernatant analysed using LC-MS Method 2.
  • Example 5 Preparation of 3,3'-((2-((6-(benzylamino)hexyl)amino)-2-((2- carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid using a nitrilase Nitrilase M2 (220 mg) was weighed into a Falcon tube and resuspended in potassium phosphate buffer (10 mL, 100 mM, pH 7.0) to a concentration of 22 mg/mL.900 ⁇ L of this solution was transferred to an Eppendorf Tube. Toluene (100 ⁇ L) was then added to the reaction mixture.
  • the reaction was started by the addition of 3,3'-((2-((6-(benzylamino)hexyl)amino)-2- ((2-cyanoethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanenitrile (4 mg) and incubating at 30 °C with shaking at 900 RPM in an Eppendorf Thermomixer. After 16 hours an aliquot (100 ⁇ L) was taken and diluted into 900 ⁇ L 1:1 ACN:H2O in an Eppendorf tube. The sample was clarified by centrifugation (10000 RPM for 1 minute) and the supernatant analysed using LC-MS Method 3.
  • Example 6 Preparation of 25,25-bis((2-carboxyethoxy)methyl)-23-oxo-1-phenyl- 5,8,11,14,17,20,27-heptaoxa-2,24-diazatriacontan-30-oic acid using a nitrilase Nitrilase M2 (220 mg) was weighed into a Falcon tube and resuspended in potassium phosphate buffer (10 mL, 100 mM, pH 7.0) to a concentration of 22 mg/mL.900 ⁇ L of this solution was transferred to an Eppendorf Tube. Toluene (100 ⁇ L) was then added to the reaction mixture.
  • the reaction was started by the addition of N-(1,3-bis(2-cyanoethoxy)-2-((2- cyanoethoxy)methyl)propan-2-yl)-1-phenyl-5,8,11,14,17,20-hexaoxa-2-azatricosan-23-amide (4 ⁇ L) and incubating at 30 °C with shaking at 900 RPM in an Eppendorf Thermomixer. After 16 hours an aliquot (100 ⁇ L) was taken and diluted into 900 ⁇ L 1:1 ACN:H2O in an Eppendorf tube. The sample was clarified by centrifugation (10000 RPM for 1 minute) and the supernatant analysed using LC-MS Method 3.

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Abstract

L'invention concerne un nouveau procédé de génération de groupes N-acétylgalactosamine (GalNAc) à l'aide d'une nitrilase et de conjugaison desdits groupes GalNAc à des oligonucléotides, tels que des oligonucléotides destinés à être utilisés en thérapie. En particulier, la nitrilase est utilisée pour catalyser la conversion d'un intermédiaire trinitrile en un intermédiaire triacide.
PCT/EP2025/063914 2024-05-23 2025-05-21 Procédé enzymatique de production de groupes de n-acétyl galactosamine Pending WO2025242702A1 (fr)

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WO1997000078A1 (fr) 1995-06-14 1997-01-03 Valio Oy Procedes de prevention ou de traitement des allergies
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