Engineered imine reductases and methods of use thereof

Abstract

Disclosed herein are engineered polypeptides having improved imine reductase (IRED) activity, the nucleotides that encode said engineered polypeptides, and methods for using said engineered polypeptides in a reduction of an imine to a chiral amine.

Description

ENGINEERED IMINE REDUCTASES AND METHODS OF USE THEREOFFIELD

[0001] This disclosure relates to engineered polypeptides having imine reductase (IRED) activity that are useful for reducing imines, polynucleotides encoding such engineered polypeptides, and uses thereof.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 664,519, filed June 26, 2024, which is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “55361_SeqListing.xml”, which was created on June 18, 2025, and is 24,382 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.BACKGROUND

[0004] Protein arginine methyltransferase 5 (PRMT5) inhibitors are useful in the treatment of MTAP-null cancers. One such inhibitor is Compound G, a second-generation PRMT5 inhibitor that targets MTA-bound state of PRMT5 in methylthioadenosine phosphorylase (MTAP)-null tumors.Initial results of the first in human study of Compound G demonstrate safety along with encouraging signs of preliminary clinical activity, and without evidence of myelosuppression, a major factor in the failure of first-generation PRMT5 inhibitors. MTAP-null cancers include pancreatic ductal adenocarcinoma, non-small cell lung cancer, cholangiocarcinoma, mesothelioma, and others, highlighting the potential utility of Compound G as a therapeutic in a variety of tumor types, based on its success in clinical trials thus far. However, more efficient methods of manufacturing are needed.SUMMARY

[0005] The present disclosure provides engineered polypeptide and polynucleotide sequences for novel biocatalysts with imine reductase (IRED) activity, and associated methods of use.

[0006] In various embodiments, the disclosure provides an engineered polypeptide comprising an amino acid sequence of SEQ ID NO: 2 and having IRED activity. In some embodiments, the polypeptide further comprises V72L, E224W, and S246I mutations. In yet other embodiments, the polypeptide further comprises a V196L mutation. In still other embodiments, the polypeptide further comprises R20Q, V52I, T130S, S195A, A254T, A263S, and G277D mutations. In other embodiments, the polypeptide further comprises R20Q, T130S, S195A, V196M, A263S, and G277D mutations. In yet other embodiments, the polypeptide further comprises R20Q, T130S, S195A, V196M, A254T, and G277D mutations. In still other embodiments, the polypeptide further comprises R20Q, T130S, S195A, and G277D mutations. The disclosure further provides engineered polypeptides having an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0007] In additional embodiments, the disclosure provides a polynucleotide encoding any one of the engineered polypeptides described herein. In various aspects, the polynucleotide comprises the sequence of SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In further aspects, the polynucleotide encoding an engineered polypeptide is at least 75%, or at least 80%, or at least 90%, or at least 95%, or at least 99% identical to any one of SEQ ID NOs: 10-16.

[0008] Further provided herein are methods of using the engineered polypeptides disclosed herein to reduce an imine to an amine, creating a chiral center in the resultant product. In some embodiments, the methods are used to prepare Compound A fromCompoundwherein the methods comprise admixing Compound B, or a salt thereof, with an engineered polypeptide as disclosed herein to form Compound A.

[0009] In various embodiments, the Compound A produced by the methods disclosed herein has an enantiomeric excess (ee) of 95%, 98%, 99%, 99.5%, or more.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows reaction time vs. product formation (Compound A) for four engineered IREDs against a comparator IRED. WT is SEQ ID NO: 1; Rd1 is SEQ ID NO: 2; Rd2 is SEQ ID NO: 3; Rd3 is SEQ ID NO: 4; and Rd4 is SEQ ID NO: 5.

[0011] Figure 2 shows lysate percent against percent conversion to product (Compound A) for four engineered IREDs against a comparator IRED. WT is SEQ ID NO: 1 ; Rd1 is SEQ ID NO: 2; Rd2 is SEQ ID NO: 3; Rd3 is SEQ ID NO: 4; and Rd4 is SEQ ID NO: 5.DETAILED DESCRIPTION

[0012] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

[0013] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0014] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.Polypeptides

[0015] The present disclosure provides engineered polypeptides having IRED activity, polynucleotides encoding the polypeptides, and methods of using the polypeptides in thepreparation of compounds useful as active pharmaceutical ingredients (API) and / or synthetic intermediates thereof. Where the description relates to polypeptides, it should also be understood that it also describes the polynucleotides encoding the polypeptides.

[0016] Disclosed herein are engineered IRED enzymes that exhibit improved efficacy for the production of a chiral amine used in the manufacture of a therapeutic compound (Compound G). The engineered enzymes of the present disclosure were modified from a commercial IRED, which has an amino acid sequence of SEQ ID NO: 1. The polypeptides of the present disclosure were engineered to have improved IRED activity for the conversion of Compound B to Compound A.

[0017] One engineered polypeptide having improved IRED activity is an amino acid sequence of SEQ ID NO: 2, having a S247P mutation, compared to SEQ ID NO: 1. Introduction of this single proline mutation in an a-helix lining the active site provides an unexpected 28x improvement in initial reaction rate for the resulting engineered polypeptide. It is hypothesized that in the prior IRED, the serine at position 247 is hydrogen-bonded to a tryptophan at position at 184, and mutation of position 247 to a serine may disrupt this interaction, creating additional space in the binding pocket. The polypeptide of SEQ ID NO: 2 was used as the starting point for additional rounds of mutations, further enhancing the reaction rates for these engineered polypeptides.

[0018] In the present disclosure, engineered polypeptides having IRED activity are described that enhance initial reaction rates when compared to the prior polypeptide of SEQ ID NO: 1. In some aspects, introduction of the disclosed mutations leads to an improvement in initial reaction rates of at least 20x, compared to that of the IRED of SEQ ID NO:1. In some cases, the improvement is at least 22x, at least 25x, at least 28x, at least 30x, at least 32x, at least 34x, at least 35x, at least 40x, at least 45x, or at least 50x.

[0019] The polypeptides as disclosed herein were developed by iterative investigation into the optimization of IRED activity. In a first round of investigation, the polypeptide of SEQ ID NO: 2 was identified, having a S247P mutation relative to SEQ ID NO:1. A second round of optimization, using the polypeptide of SEQ ID NO:2, was then initiated, and additional mutations were identified that improved the IRED activity of the Compound B to Compound A reduction. In a second round, mutations at V72L, E224W, and S246I were identified as improving the IRED activity. In a third round, a mutation at V196L was identified as improving the IRED activity. In a fourth round, mutations at (1) R20Q, V52I, T130S, S195A, A254T, A263S, and G277D; or (2) R20Q, T130S, S195A, V196M, A263S, and G277D; or (3) R20Q, T130S, S195A, V196M, A254T, and G277D; or (4) R20Q, T130S, S195A, and G277D were identified as improving the IRED activity.

[0020] In some cases, the engineered polypeptide has a sequence as recited in the below table, Table 1 , where the polypeptide of SEQ ID NO: 1 is a comparator polypeptide.TABLE 1

[0021] In some embodiments, exemplary engineered polypeptides having IRED activity possesses improved properties relative to their wild-type counterparts, and comprise the amino acid sequence of SEQ ID NO: 1 with one or more mutations introduced. In various embodiments, the mutations in the engineered polypeptide having IRED activity compriseone or more of the following: S247P, V72L, E224W, S246I, V196L, R20Q, V52I, T130S, S195A, A254T, A263S, G277D, or V196M. In some embodiments, the engineered polypeptides having I RED activity exhibit enhanced I RED activity in the conversion of Compound B to Compound A, when compared to wild-type I RED.

[0022] In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, wherein the engineered polypeptide further comprises a S247P mutation. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises a V72L, E224W, and S246I mutation. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises a V72L, E224W, S246I, and V196L mutation.

[0023] In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises a V196L mutation.

[0024] In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises R20Q, V52I, T130S, S195A, A254T, A263S, and G277D mutations. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises V72L, E224W, S246I, R20Q, V52I, T130S, S195A, A254T, A263S, and G277D mutations. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises V72L, E224W, S246I, V196L, R20Q, V52I, T130S, S195A, A254T, A263S, and G277D mutations.

[0025] In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises R20Q, T130S, S195A, V196M, A263S, and G277D mutations. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises V72L, E224W, S246I, R20Q, T130S, S195A, V196M, A263S, and G277D mutations.

[0026] In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises R20Q, T130S, S195A, V196M, A254T, and G277D mutations. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises V72L, E224W, S246I, R20Q, T130S, S195A, V196M, A254T, and G277D mutations.

[0027] In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises R20Q, T130S, S195A,and G277D mutations. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises V72L, E224W, S246I, R20Q, T130S, S195A, and G277D mutations. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises V72L, E224W, S246I, V196L R20Q, T130S, S195A, and G277D mutations. In one embodiment, the engineered polypeptide having IRED activity comprises the amino acid sequence of SEQ ID NO: 2, and further comprises V196L, R20Q, T130S, S195A, and G277D mutations.

[0028] In some embodiments, the engineered polypeptide having IRED activity comprises the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.Polynucleotides

[0029] In another embodiment, the present disclosure provides polynucleotides encoding the engineered polypeptides having IRED activity described herein. In various embodiments, the polynucleotide encodes any of the aforementioned engineered polypeptide amino acid sequences. In some embodiments, the polynucleotide encodes the engineered polypeptide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0030] In some cases, the polynucleotide has a sequence as recited in Table 2 below.TABLE 2

[0031] In some embodiments, the polynucleotide encoding the engineered polypeptide having IRED activity comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 10. The polynucleotide of SEQ ID NO: 10 encodes for the polypeptide of SEQ ID NO: 2. In some embodiments, the polynucleotideencoding the engineered polypeptide having IRED activity comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 11. The polynucleotide of SEQ ID NO: 11 encodes for the polypeptide of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding the engineered polypeptide having IRED activity comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 12. The polynucleotide of SEQ ID NO: 12 encodes for the polypeptide of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding the engineered polypeptide having IRED activity comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 13. The polynucleotide of SEQ ID NO: 13 encodes for the polypeptide of SEQ ID NO: 5. In some embodiments, the polynucleotide encoding the engineered polypeptide having IRED activity comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 14. The polynucleotide of SEQ ID NO: 14 encodes for the polypeptide of SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the engineered polypeptide having IRED activity comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 15. The polynucleotide of SEQ ID NO: 15 encodes for the polypeptide of SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the engineered polypeptide having IRED activity comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 16. The polynucleotide of SEQ ID NO: 16 encodes for the polypeptide of SEQ ID NO: 8.

[0032] In some embodiments, an isolated polynucleotide encoding any of the engineered IRED polypeptides provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides are provided as expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and / or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.Methods of Preparing Polypeptides

[0033] The engineered polypeptides having IRED activity described herein may be produced and prepared according to various methods and techniques routinely practiced in the molecular biology and / or polypeptide purification arts. Construction of an expression vector that is used for recombinantly producing a protein of interest can be accomplished using any of numerous suitable molecular biology engineering techniques known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology (2003)). To obtain efficient transcription and translation, the polynucleotide sequence in each recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operatively linked to the nucleotide sequence encoding the engineered polypeptide.

[0034] Host cells are genetically engineered with the recombinant expression vector to produce the engineered polypeptide(s) by recombinant techniques. Each of the polypeptides and fusion polypeptides described herein can be expressed in mammalian cells, yeast, bacteria, insect, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from DNA constructs. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, by Sambrook, etal., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, New York, (2001).

[0035] Generally, recombinant expression vectors useful for producing a protein of interest include origins of replication, selectable markers permitting transformation of the host cell, for example, the ampicillin resistance gene of E. coll and S. cerevisiae TRP1 gene, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. Promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.

[0036] Optionally, a heterologous sequence can be inserted in frame with the nucleotide sequence that encodes the engineered polypeptide to provide a peptide or polypeptide that imparts desired characteristics, e.g., that simplifies purification of the expressed recombinant product. Such identification peptides include a polyhistidine tag (his tag) or FLAG® epitopetag (DYKDDDDK), beta-galactosidase, alkaline phosphatase, GST, or the XPRESS™ epitope tag (DLYDDDDK; Invitrogen Life Technologies, Carlsbad, CA) and the like (see, e.g., U.S. Patent No. 5,011 ,912; Hopp et al., (Bio / Technology 6:1204 (1988)). The affinity sequence may be supplied by a vector, such as, for example, a hexa-histidine tag that is provided in pBAD / His (Invitrogen). Alternatively, the affinity sequence may be added either synthetically or engineered into the primers used to recombinantly generate the nucleic acid coding sequence (e.g., using the polymerase chain reaction).

[0037] Host cells containing described recombinant expression constructs may be genetically engineered (transduced, transformed, or transfected) with the expression constructs (for example, a cloning vector, a shuttle vector, or an expression construct). The vector or construct may be in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying encoding-nucleotide sequences. Selection and maintenance of culture conditions for particular host cells, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan. Preferably the host cell can be adapted to sustained propagation in culture to yield a cell line according to art-established methodologies for production of polypeptides. In certain embodiments, the cell line is an immortal cell line, which refers to a cell line that can be repeatedly (at least ten times while remaining viable) passaged in culture following logphase growth.

[0038] Useful bacterial expression constructs are constructed by inserting into an expression vector a structural DNA sequence encoding a desired engineered polypeptide together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The construct may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector construct and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. Any other plasmid or vector may be used as long as they are replicable and viable in the host. Thus, for example, the nucleotide sequence that encodes a protein of interest may be included in any one of a variety of a recombinant expression constructs for expressing a polypeptide. Such vectors and constructs include chromosomal, nonchromosomal, and synthetic DNA sequences, e.g., bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA; viral DNA, such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.However, any other vector may be used for preparation of a recombinant expression construct as long as it is replicable and viable in the host.

[0039] The appropriate DNA sequence(s) may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. Numerous standard techniques are described, for example, in Ausubel et al. (Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., 2003)); Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd Ed., (Cold Spring Harbor Laboratory 2001)); Maniatis et al. (Molecular Cloning, (Cold Spring Harbor Laboratory 1982)), and elsewhere.

[0040] The DNA sequence encoding an engineered polypeptide in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include LTR or SV40 promoter, the E. coll lac or trp, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Particular bacterial promoters include lacl, lacZ, T3, T5, T7, gpt, lambda PR, PL, and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruses, and mouse metallothionein-L Selection of the appropriate vector and promoter and preparation of certain recombinant expression constructs comprising at least one promoter or regulated promoter operatively linked to a nucleotide sequence that encodes an at least one engineered polypeptide is well within the level of ordinary skill in the art.

[0041] Design and selection of inducible, regulated promoters and / or tightly regulated promoters are known in the art and will depend on the particular host cell and expression system (see, e.g., E. coll arabinose operon (Pbad or Para) as described in Guzman et al., J. Bacteriology 177:4121-30 (1995); Smith et al., J. Biol. Chem. 253:6931-33 (1978); Hirsh et al., Cell 11 :545-50 (1977); PET Expression Systems (see U.S. Patent No. 4.952,496) available from Stratagene (La Jolla, CA); tet-regulated expression systems (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547-51 (1992); Gossen et al., Science 268:1766-69 (1995)); pLP-TRE2 Acceptor Vector (BD Biosciences Clontech, Palo Alto, CA) is designed for use with CLONTECH’s Creator™ Cloning Kits); see also, e.g., Sauer, Methods 14:381-92(1998); Furth, J. Mamm. Gland Biol. Neoplas. 2:373 (1997)); see, e.g., Cascio, Artif. Organs 25:529 (2001)).

[0042] The engineered polypeptide-encoding nucleic acid sequences may be cloned into a baculovirus shuttle vector, which is then recombined with a baculovirus to generate a recombinant baculovirus expression construct that is used to infect, for example, Sf9 host cells (see, e.g., Baculovirus Expression Protocols, Methods in Molecular Biology Vol. 39, Richardson, Ed. (Human Press 1995); Piwnica-Worms, “Expression of Proteins in Insect Cells Using Baculoviral Vectors,” Section II, Chapter 16 in Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., (John Wiley & Sons 1992)).

[0043] Methods that may be used for isolated and purifying an engineered polypeptide, by way of example, may include obtaining supernatants from suitable host / vector systems that secrete the engineered polypeptide into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide.

[0044] Methods for large scale production of one or more of the engineered polypeptides described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the engineered polypeptide may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.

[0045] Suitable reaction conditions under which the above-described improved properties of the engineered polypeptides carry out the desired reaction can be determined with respect to concentrations or amounts of polypeptide, substrate, co-substrate, buffer, solvent, pH, conditions including temperature and reaction time, and / or conditions with the polypeptide immobilized on a solid support, as further described below and in the Examples.

[0046] As will be appreciated by the skilled artisan, in some embodiments, one or a combination of residue differences above that is selected can be kept constant in the engineered IRED as a core feature, and additional residue differences at other residue positions incorporated into the sequence to generate additional engineered IRED polypeptides with improved properties.Methods of using the engineered polypeptides

[0047] The engineered polypeptides having IRED activity as disclosed herein are capable of converting substrates, such as converting Compound B to Compound A. In someembodiments, the engineered polypeptides disclosed herein are capable of converting Compound B to Compound A with at least 20x activity, relative to the activity of the reference polypeptide of SEQ ID NO: 1.

[0048] The reaction described herein comprises converting Compound B to Compound A via an enzymatic reduction, wherein a reduction catalyzed by an engineered polypeptide having IRED activity provides stereochemical control of the product (e.g., (S)-Compound A). Chiral control through enzyme-mediated processes are often highly selective, and in this instance, the disclosed processes provide the desired enantiomer in a stereochemical purity of greater than 99% enantiomeric excess (ee).

[0049] In addition, the disclosed processes are cost-effective when compared to conventional processes. For example, Compound A requires long lead times for synthesis of large quantities over a multi-step process. In contrast, the disclosed processes utilizing an engineered polypeptide having IRED activity provide Compound A in a much more efficient manner, as even a single amino acid substitution in the IRED sequence improves the efficiency of the process.

[0050] In various embodiments, the disclosed processes for Compound A provide advantages over conventional processes which employ numerous steps, many having poor yields, requiring the use of expensive catalysts and chiral ligands, and use of high pressures. In contrast, the disclosed processes comprise a biocatalytic reduction, avoiding the need for high pressure and which can be performed in water and at lower temperatures (e.g., 20-50 °C). In various embodiments, the biocatalytic process requires minimal unit operations - no extractions or distillations, only a pH adjustment and product filtration, resulting in fast batch cycle times. In still other embodiments, (S)-Compound A can be extracted with 2-MeTHF then distilled prior to crystallization.

[0051] In some embodiments, (S)-Compound A is a salt having the following structure:

[0052] The disclosed processes for preparing Compound A, a stereoisomer thereof, a salt thereof, or a salt of a stereoisomer thereof, comprise admixing Compound B, or a salt thereof, with an engineered polypeptide having IRED activity to form Compound A, a stereoisomer thereof, a salt thereof, or a salt of a stereoisomer thereof. In some embodiments Compound A is, or is enriched in, the (S)-stereoisomer of Compound A. By way of example, in various embodiments, in conjunction with other above or belowembodiments, (S)-Compound A produced according to the disclosed processes has an enantiomeric excess of 95% or more (e.g., 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% or more).

[0053] In various embodiments, in conjunction with other above or below embodiments, Compound A, a stereoisomer thereof, a salt thereof, or a salt of a stereoisomer thereof, is prepared in an overall yield of 75% or more, based upon Compound B. In some embodiments, Compound A, a stereoisomer thereof, a salt thereof, or a salt of a stereoisomer thereof, is prepared in a yield of 80-90%, based upon Compound B, and with a stereochemical purity of greater than 99% ee. In some embodiments, Compound A, a stereoisomer thereof, a salt thereof, or a salt of a stereoisomer thereof, is prepared in yield of 90% or more in high stereochemical purity from a compound91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more yield; +99% ee by chiral HPLC). In some embodiments, (S)-Compound A is prepared in 91% yield and +99% ee by chiral HPLC.

[0054] The engineered polypeptide having IRED activity can be any engineered polypeptide disclosed herein. The engineered polypeptide having IRED activity is present in a suitable amount. For example, in some embodiments, the engineered polypeptide having IRED activity is present in 5-10 wt%, based on Compound B. In some embodiments, the engineered polypeptide having IRED activity is present in an amount of 10 wt%, based on Compound B.

[0055] In some embodiments, the enzymatic reduction is conducted in a buffered aqueous solution. Desirably, the enzymatic reduction is conducted at a pH of 6 to 9 (e.g., a pH of 6 to 8 or 7 to 8). Suitable buffers include, for example, 2-amino-2-(hydroxymethyl)-1 ,3- propanediol (Tris) and phosphate buffers. In some embodiments the buffer is a potassium phosphate buffer (pH 7.4) present in an amount of 30 volumes.

[0056] The enzymatic reaction mixture comprises any suitable reductant, oxidant, and / or co-factors capable of maintaining enzymatic activity at a desired rate. By way of example, in some embodiments, the admixing of Compound B, or a salt thereof, with an engineered IRED is conducted using nicotinamide adenine dinucleotide phosphate (NADP+) (3 wt%), glucose dehydrogenase (GDH) (3 wt%), and glucose (reductant). In some embodiments, a slight excess of NADP+ (1.01 mmol) based upon the substrate (i.e. , Compound B).Similarly, an excess of the reductant can be used (e.g., 1.1 eq, 1.2 eq, 1.3 eq, 1.4 eq, or 1.5eq reductant). In some embodiments, the enzymatic reaction mixture comprises 1.4 eq. of D-(+)-glucose.

[0057] The admixing of Compound B, or a salt thereof, with an engineered IRED is conducted at a suitable temperature. In various cases, the admixing reaction is conducted at a temperature of less than 50 °C (e.g., 45 °C). For example, in some embodiments, the admixing of Compound B, or a salt thereof, with an IRED is conducted at 20-45 °C, 20-40 °C, 20-35 °C, or 30-35 °C.EXAMPLES

[0058] The following examples further illustrate the disclosed process, but of course, should not be construed as in any way limiting their scope.Example 1. Gene optimization and synthesis.

[0059] The gene encoding the wild-type IRED was codon-optimized for heterologous expression in Escherichia coli. The codon-optimized DNA sequence was synthesized as an insert within the pET28a(+) vector between the BamHI and Hindlll restriction sites. This starting construct served as the template for all modifications described herein. Based on this design, the various recombinant IREDs were all produced in BL21(DE3) E. coli cells with an N-terminal polyhistidine tag.Example 2. High-throughput generation of protein libraries.

[0060] DNA libraries were generated starting from the initial codon-optimized DNA construct using well-established molecular biology techniques, including (1) site-saturation mutagenesis, (2) site-directed mutagenesis, and (3) multiple site-directed mutagenesis to recombine beneficial mutations that were identified throughout the evolution campaign. Chemically competent BL21(DE3) E. coli cells were transformed with the resulting DNA library, resulting in each individual colony harboring a uniquely mutated gene.

[0061] The individual colonies were used to inoculate 800-pL cultures of Luria-Bertani broth containing 50 pg / mL kanamycin. In plate format, the cultures were incubated overnight at 37 °C and 80% humidity, with shaking at 1000 rpm. A portion of the overnight cultures (30 pL) were used to inoculate 400-pL cultures of Terrific Broth containing 50 pg / mL kanamycin. In plate format, the cultures were incubated overnight at 37 °C and 80% humidity, with shaking at 1000 rpm. After the desired cell density was reached (OD600 of 0.8-1.5), the expression was induced with the addition of isopropyl IS-D-1 -thiogalactopyranoside to a final concentration of 0.5 mM. In plate format, the expression cultures were incubated overnight at 18 °C and 80% humidity, with shaking at 1000 rpm. After 20 hours, the plates werecentrifuged at 3500 rpm for 5 minutes. The supernatant was discarded and the plates containing the remaining cell pellets containing recombinant protein were stored at -80 °C.

[0062] Prior to lysing the cells, 96-well plates containing cell pellets were thawed at room temperature. The thawed cells were resuspended in lysis buffer (100 mM potassium phosphate pH 7.5, 2 mg / mL lysozyme, 10 U / mL benzonase, 0.1 mM phenylmethylsulfonyl fluoride) and incubated at 22 °C with shaking at 1000 rpm. After 2 hours, the plates were centrifuged at 3500 rpm for 5 minutes. The supernatant (cleared lysate containing soluble proteins) was used directly in high-throughput reactions.Example 3. High-throughput biocatalvtic reactions.

[0063] To set up high-throughput reactions, 10 pL of fresh cleared lysate containing soluble proteins was transferred to fresh 96-well plates. A total of 190 pL reaction mix was added so that the final reaction components were as follows: 100 mM potassium phosphate pH 8, 5 mM substrate (5-(4-(trifluoromethyl)phenyl)-3,6-dihydro-2H-1,4-oxazine - Compound B), 5% (v / v) dimethyl sulfoxide, 1.4 equivalents glucose, 10% (w / w) glucose dehydrogenase, 10% (w / w) nicotinamide adenine dinucleotide phosphate (NADP). Reactions were incubated at 30 °C, 600 rpm. After 20 hours, reactions were quenched with 800 pL aceton itrile / water (80:20) containing 0.1% (v / v) trifluoroacetic acid. Samples were filtered with 96-well filter plate (0.4 pm) by centrifugation. Resulting filtrates were analyzed by high-throughput mass spectrometry by bypassing the column compartment and performing overlapping injections, giving an injection rate of 2 injections per minute (Table 3, Method #1). Activity of each variant was defined as fold improvement in product formation (Compound A) over the template sequence (SEQ ID NO: 1), see Table 4 and Figures 1 and 2. Variants with activity above a certain threshold were analyzed by chiral high-performance liquid chromatography (Table 3, Method #2).

[0064] Trends in activity and selectivity data were correlated to the key mutations added in each variant; these data were used to determine the template sequence and mutagenesis strategy for the next round. In each round of evolution, the same general protocol for library generation and reactions was followed. However, slight modifications to each round were made, including (1) changing buffer pH and molarity, (2) increasing substrate concentration, (3) removal of DMSO from reaction, and (4) pre-incubation of the cleared lysate at elevated temperatures to test the thermal stability of the protein variants.TABLE 3Method #1massFIA-IInstrument Agilent 1290 Infinity II paired with Agilent MSDMobile Phase 90% acetonitrile, 10% water, 0.02% formic acid (isocratic)Flow rate 0.5 mL / minColumn temperature Not controlled Run time Continuous Injection volume 1 uL Capillary voltage 4.0 kV Fragmenter value 110 Drying gas flow rate 12 L / min Nebulizer pressure 45 psi Drying gas temp 350 C Vaporizer temp 130 C SIM mass 232.2 m / zMethod #2 (chiral analysis)Instrument column CHIRALPAK® IH-3, 100 x 4.6 mm, 3 pmMobile Phase 100% ethanol (isocratic)Flow rate 1.0 mL / minRun time 8 minColumn temperature 30 °CInjection volume 0.5 pLWavelength 230 nm

[0065] The results of the reactions with different engineered IREDs are shown in Table 4 below, compared to that of SEQ ID NO: 1.TABLE 4Enzyme . ... . ...’ ._ initial melting . .. .. .(SEQ ID . . „amutations NotesNO)ate temp. ..n orwild-type Prozomix enzyme (IRED-1'43 9 C' 0712-C)2 28x 302 °C S247P destabilizing proline mutation in alpha-helix o _ .q{-7„rV72L, E224W, S246I, +5.5 °C improvement in thermal3J4x JOV uS247PStabilityA 99 An 1 °r V72L, V196L, E224W, +4.4 °C improvement in thermal4zzx 4U.1 u S246I, S247P stabilityR20Q, V52I, V72L, c Rn °r T130S, S195A, V196L, +5.4 °C improvement in thermal o ouxu E224W, S246I, S247P, stabilityA254T, A263S, G277D

[0066] Initial reaction rates are also shown in Figure 1 , over the first 30 minutes of reaction time, and each engineered polypeptide enzyme has vastly improved rate over that of that of SEQ ID NO: 1.

[0067] Enzyme loading impact was also investigated, and the results are shown in Figure 2, where lower loading amounts of enzyme resulted in much higher conversion rates to Compound A, compared to that of the comparator enzyme of SEQ ID NO: 1.Example 4. Large-scale demonstration with engineered IRED.

[0068] To a 5-L jacketed reactor set to 20 °C, tert-butyl N-[2-[2-oxo-2-[4- (trifluoromethyl)phenyl]ethoxy]ethyl]carbamate (200 g, 0.576 mol, 1.0 equiv) and dimethyl sulfoxide (545 mL, 7.68 mol, 13.3 equiv) were added. The reactor temperature was increased to 40 °C. Hydrochloric acid (2.59 L of 1 M solution, 4.50 equiv) was dosed in over 90 minutes. The reactor temperature was increased to 60 °C. Vacuum cycles were performed by pulling vacuum at 500 torr for 5 minutes every 30 minutes. After 3 hours, the reactor temperature was decreased to 0 °C to rapidly cool the reaction, followed by polish filtration of the homogeneous solution. In a reverse quench, the filtered reaction was slowly added (over 2 hours) back to the reactor containing sodium carbonate (2.00 L of 1 M solution, 3.50 equiv). After holding at 0 °C overnight, the reactor temperature was increased to 20 °C and the pale-yellow slurry was filtered. The wet cake was washed with water / DMSO (9:1 ; 3 volumes), followed by water washes (2 x 3 volumes).

[0069] The washed solids were charged into the reactor containing potassium phosphate buffer pH 8 (400 mM, 2 L total). The following components were charged: glucose (5.8 g, 32 mmol, 1.4 equiv.), nicotinamide adenine dinucleotide phosphate (1.98 g, 0.99 wt%), glucose dehydrogenase (0.26 g, 0.13 wt%), and IRED variant (4.0 g, 2.0 wt%). The reactor temperature was increased to 30 °C. After 4 hours, only 0.77% of imine substrate Compound B was observable by liquid chromatography and the pH of the reaction had levelled off. The pH of the reaction was adjusted to pH 7.3 with potassium hydroxide (200 mL of 2 M solution). Celite (0.66 g, 0.33 wt%) was charged to the reactor, followed by 2- MeTHF (6.6 volumes). The reactor temperature was increased to 40 °C and left overnight. The celite was removed by filtration and washed with of 2-MeTHF (1.5 volumes). The biphasic filtrate was returned to the reactor and the aqueous layer was removed. The organics were washed with aqueous sodium chloride (20 wt%, 2 volumes). The organic layer was distilled down to 2 volumes (45 °C, 150 torr). An additional 6.6 volumes of 2-MeTHF were charged. The solution was polish filtered and washed with 1.5 volumes 2-MeTHF.

[0070] To initiate crystallization, hydrochloric acid (5.2 M solution in isopropanol, 11 mL, 0.10 equiv) was charged over 20 minutes. The solution was seeded with addition of the hydrochloride salt of (S)-3-(4-(trifluoromethyl)phenyl)morpholine Compound A (0.50 g, 0.25 wt%). An additional amount of hydrochloric acid (5.2 M solution in isopropanol, 100 mL, 0.90 equiv) was charged over 160 minutes. The crystallization was aged overnight. The slurrywas filtered, and the solids were washed twice with 2-MeTHF (2 volumes). The resulting solids were dried under nitrogen and vacuum overnight, providing 132 g of (S)-3-(4- (trifluoromethyl)phenyl)morpholine Compound A (83.8% potency adjusted yield, >99.9% enantioselectivity, 97.8 wt% purity, 0.02 wt% residual protein).

Claims

What is claimed is:1 . An engineered polypeptide comprising an amino acid sequence of SEQ ID NO: 2 and having imine reductase (IRED) activity.

2. The engineered polypeptide of claim 1 , further comprising V72L, E224W, and S246I mutations.

3. The engineered polypeptide of claim 1 or 2, further comprising a V196L mutation.

4. The engineered polypeptide of any one of claims 1-3, further comprising R20Q, V52I, T130S, S195A, A254T, A263S, and G277D mutations.

5. The engineered polypeptide of claim 1 or 2, further comprising R20Q, T130S, S195A, V196M, A263S, and G277D mutations.

6. The engineered polypeptide of claim 1 or 2, further comprising R20Q, T130S, S195A, V196M, A254T, and G277D mutations.

7. The engineered polypeptide of any one of claims 1-3, further comprising R20Q, T130S, S195A, and G277D mutations.

8. The engineered polypeptide of any one of claims 2-7 having an amino acid sequence of any one of SEQ ID NOs: 3-8.

9. A polynucleotide encoding the engineered polypeptide of any one of claims 1 -8.

10. The polynucleotide of claim 9 having a nucleotide sequence that is at least 75% identical to any one of SEQ ID NOs: 10-16.11 . The polynucleotide of claim 10, having a nucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 10-16.

12. The polynucleotide of claim 11 , having a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 10-16.

13. The polynucleotide of claim 12, having a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 10-16.

14. The polynucleotide of claim 13, having a nucleotide sequence that is at least 99% identical to any one of SEQ ID NOs: 10-16.

15. A method of preparing Compound A from Compound Bcomprising admixing Compound B, or salt thereof, with the engineered polypeptide of any one of claims 1-8 to form Compound A.

16. The method of claim 15, wherein Compound A has an enantiomeric excess of 95% or more.

17. The method of claim 16, wherein Compound A has an enantiomeric excess of 98% or more.

18. The method of claim 17, wherein Compound A has an enantiomeric excess of 99% or more.

19. The method of claim 18, wherein Compound A has an enantiomeric excess of 99.5% or more.

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