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WO2004113524A1 - Variants d'aminoacylase, acides nucleiques les codant, et utilisations associees - Google Patents

Variants d'aminoacylase, acides nucleiques les codant, et utilisations associees Download PDF

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WO2004113524A1
WO2004113524A1 PCT/CA2004/000938 CA2004000938W WO2004113524A1 WO 2004113524 A1 WO2004113524 A1 WO 2004113524A1 CA 2004000938 W CA2004000938 W CA 2004000938W WO 2004113524 A1 WO2004113524 A1 WO 2004113524A1
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variant
aminoacylase
seq
amino acid
cpg2
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Holger A. Lindner
Robert Menard
Traian Sulea
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National Research Council of Canada
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the cytosolic enzyme aminoacylase, and more particularly to variants thereof, and nucleic acids coding same.
  • the present invention also relates to the use of said polypeptides and nucleic acids in directed enzyme prodrug therapy (DEPT).
  • DEPT directed enzyme prodrug therapy
  • the high systemic toxicity is the major drawback of chemotherapy for the treatment of cancer.
  • one strategy to overcome this problem and to deliver high drug concentrations to the target cell is cancer tissue specific drug activation by an exogenous enzyme. Suitable enzymes exhibit a catalytic activity normally not found in the human body.
  • a low molecular weight precursor of a chemotherapeutic drug, a so- called prodrug is administrated and the enzyme converts it into its active form, which kills surrounding tumor cells while other tissues are spared from toxicity.
  • DEPT directed enzyme prodrug therapy '
  • ADPT antibody- or gene-directed enzyme prodrug therapy
  • GDEPT antibody- or gene-directed enzyme prodrug therapy
  • CPG2 is capable of catalytically liberating a range of benzoic acid, phenol, and aniline nitrogen mustards from comparatively non-toxic prodrugs.
  • the alkylating activity of the mustard portion is masked by conjugation of the benzoic acid, phenol or aniline ring, respectively, to the amino group of a glutamic acid residue that can be cleaved off by CPG2.
  • the prodrugs are at least 100-fold less reactive.
  • the anti-enzyme immune response is at the same time directed against the tumor cells expressing the foreign enzyme.
  • results from animal models using the Herpes simplex thymidine kinase ganciclovir- enzyme prodrug system indicated that the immune system not only attacked transgene expressing tumor cells, but was also sensitized toward the respective tumor in general. However, it has to be assumed that after the first application the immune response will rapidly neutralize the expression of the transgene, i.e. the foreign enzyme. As a result, there will be no efficient prodrug activation during repeated rounds of treatment.
  • the present invention concerns the characterization of variants of aminoacylase, more particularly, the variants of the human aminoacylase 1 (Acy 1), and their use in directed enzyme prodrug therapy (DEPT).
  • Acy 1 human aminoacylase 1
  • DEPT directed enzyme prodrug therapy
  • an object of the present invention is to provide a variant of an aminoacylase, wherein said variant has an increased substrate catalytic specificity and has an amino acid sequence at least 85% identical to the SEQ ID no. 1.
  • Another object of the present invention is to provide an isolated nucleic acid molecule that encodes for a variant of an aminoacylase as defined above and vectors containing the isolated nucleic acid molecule.
  • Another object of the present invention concerns a process for producing a variant of an aminoacylase as defined above, the process comprising the steps of : a. culturing a host cell containing a vector as defined in claim 11 or 12 under condition sufficient for the production of said non-immunogenic variant of an aminoacylase; and b. recovering said variant of an aminoacylase.
  • Figure 1 shows a ribbon diagram of the zinc-binding domain in a variant of an aminoacylase according to a preferred embodiment of the invention, namely the T347G mutant of hAcyl (PDB ID 1Q7L).
  • Glycine was modeled in place of a putative L-norleucine ligand molecule, and is shown in ball-and-stick representation.
  • Zinc ions are represented as gray spheres.
  • Figure 2 shows a stereo close-up view of the superposition of the zinc centers in the T347G mutant of hAcyl (PDB ID 1Q7L) in complex with glycine, Pseudomonas sp. CPG2 (PDB ID 1CG2) and S. typhimu um PepT (PDB ID 1FNO) (both without ligand), L delbrueckii PepV (PDB ID 1LFW), AAP (PDB ID 1AMP), and SGAP (PDB ID 1QQ9) in complex with Asp ⁇ [PO 2 CH 2 ]AlaOH, L-leucinephosphonic acid and methionine, respectively.
  • the structures are colored in red, green, brown, cyan, blue and purple for hAcyl, CPG2, PepT, PepV, AAP and SGAP, respectively. Only amino acid side chains are shown. The numbering is given for hAcyl .
  • zinc 2 was taken as the first, and the zinc 1 as the second fixed point, followed by alignment with the O ⁇ atom of Asp113.
  • Figure 3 shows the enzymatic properties of a preferred aminoacylase variant, namely T347G.
  • Benzoyl-amino acids and derived compounds are analogs of early generation CPG2-activated prodrugs.
  • the phenylpropionyl-moiety partly mimicks the drug portion in CPA-activated prodrugs.
  • Wild-type hAcyl generally hydrolyzes acetylamino acids about 100-fold more efficient than benzoylamino acids.
  • a The bar chart illustrates that the T347G mutant of hAcyl has virtually lost the ability to distinguish between acetyl- and benzoylamino acids.
  • the mutant enzyme shows an absolute improvement toward benzoylamino acid substrates.
  • the changes are mainly due to increased / ca t values.
  • the k ca t/KM values for selected para-amino substituted benzoylamino acid substrates reflect changes in mainly k ca t, while KM values remained largely unaffected by the mutation (not shown).
  • Figure 4 shows the structures of the small domains of enzymes from the Acy1/M20 family.
  • A topology diagram for the lid domain in L delbrueckii PepV and the dimerization domains from both monomers in Pseudomonas sp. CPG2.
  • Subdomain 1 (gray) and 2 (white) of PepV show apparent similarity. However, strand 8 and 12 are only found in subdomain 1 , and strands 3 and 7 only in subdomain 2.
  • the ⁇ -sheet composed of the latter two strands is also present in the dimerization domain of CPG2.
  • ⁇ backbone trace superposition of subdomain 1 and 2 in the lid domain of PepV (blue) and the two associated dimerization domains in CPG2 (red and green).
  • Known active site residues in PepV are shown in stick representation, from left to right, Arg350, Asn217 (both carboxy terminal docking), and His269 (transition state stabilization).
  • Corresponding residues from CPG2 are also shown. The enlargement above additionally shows the corresponding residues in PepT. Arg288 from CPG2 (red), and Arg280 from PepT (yellow) reside in the monomer, which superimposes with subdomain 1 of PepV. Asn275 and His229 from CPG2 (green), and His223 in PepT (purple) are recruited from the opposite monomer which superimposes with subdomain 2 of PepV.
  • the side chain of His229 shows a ⁇ , rotation by about 90 degrees relative to the other two structures, and coordinates an additional inter-dimeric zinc ion in the protein crystal (not shown).
  • FIG. 5 shows the enzyme complementation assay I.
  • N and D indicate the asparagine and aspartic acid mutations in these two positions.
  • H206N was mixed with mutants of the zinc- binding domain of hAcyl to yield total protein concentrations of 150 ⁇ g/ml. After an equilibration period, samples were assayed for N-acetyl- L-methionine (10 mM) hydrolyzing activity for 20 min. ⁇ , time dependence of activity reappearance after mixing of H206N and E147D in equal amounts.
  • Figure 6 shows the enzymatic properties of preferred variants of aminoacylase of the invention, namely T347S, F187S, M211 and I177A.
  • Benzoyl-amino acids and derived compounds are analogs of early generation CPG2-activated prodrugs.
  • the phenylpropionyl-moiety partly mimicks the drug portion in CPA-activated prodrugs.
  • Catalytic efficacies are given as k c Ku values in percentage of the wild-type values given in Figure 3A.
  • Figure 7 shows putative active site residues in the Acy1/M20 family: A schematic representation.
  • the metal-binding domains are shown in red, and the dimerization domains of the opposing subunits in blue and green, respectively.
  • the scissile bond in the acyl-amino acid is printed in red.
  • R1 represents an amino acid side chain, and R2 defines the acyl moiety in the substrate.
  • Figure 8 shows the enzyme complementation assay II.
  • mutants were equilibrated and assayed for N- acetyl-L-methionine hydrolyzing activity.
  • Figure 9 shows the conformational switch during catalysis in the Acy1/M20 family: A molecular view provided by the structures of CPG2 and PepV.
  • Figure 10 shows that the catalytically-competent substrate-binding site is located at the junction between three domains: One zinc-binding domain and two interacting dimerization domains
  • Figure 11 shows the amino acid sequence of the wild type human aminoacylase 1 (Acy 1) and identified as SEQ ID NO. 1.
  • Figure 12 shows the amino acid sequence of a variant of an aminoacylase according to a preferred embodiment of the invention, namely variant I177A, and identified as SEQ ID NO.2.
  • Figure 13 shows the amino acid sequence of a variant of an aminoacylase according to a preferred embodiment of the invention, namely variant
  • Figure 14 shows the amino acid sequence of a variant of an aminoacylase according to a preferred embodiment of the invention, namely variant M2111, and identified as SEQ ID NO.4.
  • Figure 15 shows the amino acid sequence of a variant of an aminoacylase according to a preferred embodiment of the invention, namely variant T347G, and identified as SEQ ID NO.5.
  • Figure 16 shows the amino acid sequence of a variant of an aminoacylase according to a preferred embodiment of the invention, namely variant
  • T347S T347S, and identified as SEQ ID NO.6.
  • Figure 17 shows the nucleotide sequence of the gene encoding the wild type human aminoacylase 1 (Acy 1), and identified as SEQ ID NO.7.
  • Figure 18 shows the nucleotide sequence encoding the variant of figure 12, and identified as SEQ ID NO.8.
  • Figure 19 shows the nucleotide sequence encoding the variant of figure 13, and identified as SEQ ID NO.9.
  • Figure 20 shows the nucleotide sequence encoding the variant of figure 14, and identified as SEQ ID NO.10.
  • Figure 21 shows the nucleotide sequence encoding the variant of figure 15, and identified as SEQ ID NO.11.
  • Figure 22 shows the nucleotide sequence encoding the variant of figure 16, and identified as SEQ ID NO.12. Detailed description of the invention
  • the present invention relates to the cytosolic enzyme aminoacylase, and more particularly to variants thereof, and nucleic acids coding same.
  • the present invention also relates to the use of said variants and nucleic acids in directed enzyme prodrug therapy (DEPT), such as antibody directed enzyme prodrug therapy (ADEPT) or gene directed enzyme prodrug therapy (GDEPT).
  • DEPT directed enzyme prodrug therapy
  • ADPT antibody directed enzyme prodrug therapy
  • GDEPT gene directed enzyme prodrug therapy
  • the present invention concerns the use of a variant of an aminoacylase or a functional derivative thereof, wherein said variant has an increased substrate catalytic specificity and has an amino acid sequence at least 85% identical, and preferably at least 90% identity, or even preferably 95% identity to part or all of SEQ ID NO:1 as shown in Fig. 11.
  • the term "increased substrate catalytic specificity” refers to an enhanced level of measurable catalytic specificity of efficacy of a polypeptide, such as an aminoacylase variant according to the invention toward a substrate (such as benzoyl - and/or phenylpropionyl-amino acids prodrug-like substrates) in a given assay relative to the measurable level of catalytic specificity of a wild-type aminoacylase.
  • the variant comprises a mutation which consists of a substitution of an amino acid residue at a position selected from the group consisting of 347, 211, 187 and 177 as set forth in SEQ ID no 1. More preferably, the variant comprises a mutation selected from the group consisting of I177A, F187S, M211I, T347G, and T347S, in the amino acid sequence shown in SEQ ID no 1.
  • a mutation of I177A, F187S, M211I, T347G, and T347S means that a substitution of amino acid residue He to Ala, Phe to Ser, Met to lie, Thr to Gly and Thr to Ser occurred respectively in positions 177, 187, 211, 347 and 347 of SEQ ID No l .
  • the variant of the invention comprises an amino acid sequence selected form the group consisting of SEQ ID Nos: 2, 3, 4, 5, and 6, as shown in Figs 12 to 16.
  • the variant of the invention preferably consists of a variant of a human aminoacylase 1 (hAcy 1) polypeptide.
  • hAcy 1 human aminoacylase 1
  • the T347G, T347S, M211 I, F187S and I177A mutants of hAcyl themselves present non-limiting examples of changing enzyme substrate specificity toward a desired direction, i.e. against prodrug-like molecules resembling, for instance, to CPG2- or CPA-activated prodrug (see Figures 3 and 6).
  • the principle modes are the amino side chain size reductions and changes in side chain shape.
  • This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or homology for an optimal alignment.
  • a program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
  • polypeptide refers to any peptide, or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post- translational modifications, but also by chemical modification techniques.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, selenoylation, sulfation and transfer-RNA mediated addition of amino acids to proteins
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well. With respect to protein or polypeptide, the term "isolated polypeptide" or
  • isolated and purified polypeptide is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated polynucleotide molecule contemplated by invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e. g., nucleic acid, oligonucleotide, protein, polypeptide, peptide, etc.). More preferably, the preparation comprises at least 75%» by weight, and most preferably 90-99% by weight, the compound of interest.
  • the compound of interest e. g., nucleic acid, oligonucleotide, protein, polypeptide, peptide, etc.
  • Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • the present invention concerns the use of an isolated or purified nucleic acid molecule encoding an aminoacylase variant of the invention. Therefore, the nucleic acid molecule of the invention has preferably a nucleotide sequence which is at least 75% identical, more particularly 80% identical and even more particularly 95% identical to part or all of SEQ ID No. 7 and functional fragments thereof, as shown in figure 17. More preferably, the variant comprises a nucleotide sequence selected from the group consisting of SEQ ID No. 8, 9, 10, 11, and 12, and functional fragments thereof, as shown in figures 18 to 22.
  • a “functional fragment”, as is generally understood and used herein, refers to a nucleic acid sequence that encodes for a functional biological activity that is substantially similar to the biological activity of the whole nucleic acid sequence. In other words, it refers to a nucleic acid or fragment(s) thereof that substantially retains the capacity of encoding for an aminoacylase variant of the present invention.
  • fragment refers to a nucleic acid sequence (e.g., cDNA) which is an isolated portion of the subject nucleic acid constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art.
  • cDNA nucleic acid sequence
  • isolated nucleic acid molecule refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived.
  • the "isolated nucleic acid molecule” may comprise a DNA molecule inserted into a vector, such as a plasmid, a bacteriophage or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote.
  • An “isolated nucleic acid molecule” may also comprise a cDNA molecule.
  • Amino acid or nucleotide sequence “identity” and “similarity” are determined from an optimal global alignment between the two sequences being compared. An optimal global alignment is achieved using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453). "Identity” means that an amino acid or nucleotide at a particular position in a first polypeptide or polynucleotide is identical to a corresponding amino acid or nucleotide in a second polypeptide or polynucleotide that is in an optimal global alignment with the first polypeptide or polynucleotide. In contrast to identity, "similarity" encompasses amino acids that are conservative substitutions.
  • a “conservative” substitution is any substitution that has a positive score in the blosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919).
  • sequence A is n% similar to sequence B
  • sequence B is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides and conservative substitutions.
  • sequence A is n% identical to sequence B is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides.
  • polynucleotide(s) generally refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • This definition includes, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, cDNA, single- and doublestranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. "Polynucleotide(s)" embraces short polynucleotides or fragments comprising at least 6 nucleotides often referred to as oligonucleotide(s).
  • polynucleotide(s) as it is employed herein thus embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells which exhibits the same biological function as the polypeptide encoded by any one of SEQ ID NOS.1 to 4.
  • polynucleotide(s) also embraces short nucleotides or fragments, often referred to as “oligonucleotides”, that due to mutagenesis are not 100% identical but nevertheless code for the same amino acid sequence.
  • the invention is further directed to a vector comprising a nucleic acid molecule as defined above, and more particularly directed to a cloning or expression vector which is capable of directing expression of the polypeptide encoded by the nucleic acid molecule in a vector-containing cell.
  • vector refers to a polynucleotide construct designed for transduction/transfection of one or more cell types.
  • Vectors may be, for example, "cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors” which are designed for expression of a nucleotide sequence in a host cell, or a “viral vector” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors", which comprise the attributes of more than one type of vector.
  • vectors suitable for stable transfection of cells and bacteria are available to the public (e.g. plasmids, adenoviruses, baculoviruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any ofthe polynucleotide molecules ofthe invention.
  • the present invention provides a process for producing a variant of an aminoacylase as defined above, the process comprising the steps of : a. culturing a host cell containing a vector as defined above under condition sufficient for the production of said non-immunogenic variant of an aminoacylase; and b. recovering said variant of an aminoacylase.
  • the host cell may be any type of cell (a transiently-transfected mammalian cell line, an isolated primary cell, or insect cell, yeast (Saccharomyces cerevisiae, Pichia pastoris), plant cell, microorganism, or a bacterium (such as E. coli). More preferably the host is an insect cell, such as Sf21.
  • Human aminoacylase 1 (hAcyl) as a potential prodrug activating enzyme
  • the present invention generally relates to strategies that tackle the problem of immunogenicity of prodrug-activating enzymes, and considers the closest human homolog of CPG2, the cytosolic enzyme aminoacylase 1 (Acyl), as an alternative prodrug activating enzyme.
  • CPG2 human Acyl (hAcyl) meets a number of requirements, and shows some intrinsic benefits for use in ADEPT and GDEPT.
  • HAcyl can be categorized as a stable protein. Its homodimeric structure would benefit binding avidity after coupling/fusion to a targeting agent. Minute amounts ofthe enzyme are known to occur in the blood after liver cell damage, but no anti-hAcyl immune response has yet been described. Hence, we do not anticipate ectopic application of a hAcyl variant to bear an elevated risk, when compared to any other human protein with limited recombinant modifications.
  • the inventors explored the potential of hAcyl to be used in enzyme prodrug activation.
  • CPG2 and hAcyl both exhibit amino acid deacylating activities with major differences toward the P1 -moiety of the substrate, which actually features the nitrogen mustard drug portion in CPG2-activated prodrugs.
  • the preference of wild-type hAcyl for small acyl-moieties precludes the acceptance of CPG2- prodrug substrates.
  • the inventors have delineated the active site architecture and putative catalytic mechanism of Acy1/CPG2 family enzymes, using mutational analyses of hAcyl and structural analyses, including the determination of the 3D- structure of the Zn-binding domain of a hAcyl variant.
  • the inventors have furthermore identified determinants of hAcyl S1-subsite substrate specificity.
  • the inventors were able to design single amino acid mutants in four distinct positions of the S1-subsite in hAcyl that led to 5 - 200fold improvements in catalytic efficacies toward CPG2-prodrug-like substrates. This may present a first step in the engineering of hAcyl towards prodrug activation.
  • a group of nitrogen mustard prodrugs had been developed for activation by human carboxypeptidase A (CPA) (Smith et al., 2001). They are mechanistically similar, but structurally somewhat distinct from CPG2-activated prodrugs.
  • CPA carboxypeptidase A
  • CPA-activated prodrugs may be regarded as a second lead for prodrugs, which may be activated by an Acyl -variant.
  • this example emphasizes how a common chemical mechanism of action for masking the toxicity of nitrogen mustards still allows structural variability of the prodrug.
  • CPG2 and Acyl are assigned to the M20 family of clan MH (http://merops.sanger.ac.uk) (Rawlings et al., 2002).
  • the MEROPS classification of peptidases groups peptidases with significant sequence similarity into a family, and assigns families of common evolutionary origin to a clan.
  • Wouters and Husain pointed out that members of the MH and MF clans of di-zinc peptidases, together with the MC clan of mono-zinc peptidases display three different catalytic zinc centers that have evolved in a similar structural scaffold, which is exemplified by carboxypeptidase A of clan MC.
  • Acyl plays a general role in the cytosolic breakdown of N ⁇ - acetylated amino acids (Gade and Brown, 1981) generated during protein degradation (Jones et al., 1991). Besides other functional aminoacylase enzymes (Boyen et al., 1992), the Acy1/M20 family contains several enzymes that are known as exopeptidases including CPG2 (Levy and Goldman, 1967; Goldman and Levy, 1967; Sherwood et al., 1985), PepT and the dipeptidase enzyme PepV from various bacterial sources (Barret et al., 1998; Hellendorn etal., 1997).
  • Enzymes of the Acy1/M20 family have shown potential for different applications.
  • biocatalysis the high stereoselectivity of Acyl allows the preparation of L-amino acids from racemic mixtures of N-acyl-L-amino acids.
  • CPG2 is being considered as therapeutic agent in ADEPT for cancer treatment (Burke, 2000).
  • AAP Aeromonas proteolytica aminopeptidase
  • SGAP Streptomyces griseus aminopeptidase
  • CPG2 The crystal structures of two Acyl homologs, CPG2 (Rowsell et al., 1997), and PepT from Salmonella typhimurium (Hakansson and Miller, 2002), are each folded into a metal-binding domain, and a smaller dimerization domain which is inserted in the middle of the sequence of the metal-binding domain. In the structures of both enzymes, the two domains display an open conformation.
  • the metal-binding domains in CPG2 and PepT exhibit high structural similarity to the two single-domain proteins AAP and SGAP, and are thought to be responsible for catalysis.
  • PepV The structure of PepV was determined in a complex with an inhibitor, Asp ⁇ [PO 2 CH 2 ]AlaOH, which mimics the transition state of a dipeptide substrate, bound in a hydrophobic cavity between the zinc- binding and the lid domain. Its interactions with the enzyme pinpoint the active site residues. In addition to residues from the zinc-binding domain, residues from the lid domain also appear to be involved in substrate-binding and catalysis. Both zinc sites, hAcyl and a conserved glutamate in their immediate vicinity are essential for catalysis. The lid domain in the related, but monomeric enzyme PepV mimics the structure of a dimer, and thereby inserts a catalytic histidine into the active site.
  • an inhibitor Asp ⁇ [PO 2 CH 2 ]AlaOH
  • Acy1/M20 family enzymes In contrast to the single-domain enzymes from the M28 family, Acy1/M20 family enzymes generally contain a second, smaller domain. As mentioned above, most of these enzymes exist as homodimers. In CPG2 and S. typhimurium PepT, the small domains were shown to mediate enzyme dimerization through side-by- side packing of their 4-stranded ⁇ -sheets thereby forming a contiguous extended 8-stranded sheet. In contrast, L. delbrueckii PepV exists in a monomeric form. The crystal structure of this enzyme revealed that its small domain contains a central eight-stranded ?-sheet. This domain can be subdivided into two subdomains, each composed of six ⁇ -strands.
  • Subdomain 1 encompasses strands 1 , 8, 9, 10, 11 , 12 and helices III and IV, while subdomain 2 includes strands 2, 3, 4, 5, 6, 7 and helices I and II (Fig. 4).
  • Subdomain 2 has the same topology as the dimerization domain of CPG2 (Fig. 4A) and of PepT.
  • the two subdomains in the lid domain of PepV mimic the arrangement of the two dimerization domains within the CPG2 and PepT dimers.
  • the superposition of the entire small domain of PepV on the small domains of the CPG2 dimer (Fig. 4B) overlaps 196 Cor atoms out of 230 with a rms deviation of 1.54 A.
  • His269 was assigned a role in transition state stabilization, and, Arg350 and Asn217 in anchoring the free C-terminus of the substrate. These three residues were found to be conserved in 98%, 89% and 72% of Acy1/M20 family sequences, respectively (http://merops.sanger.ac.uk ). Beyond that, there is no identifiable sequence identity between subdomain 1 and 2 in PepV, and any of the dimerization domains compared here (Fig. 4C), which reinforces the functional significance ofthe histidine, arginine and, possibly, asparagine conservations.
  • the role of a conserved histidine in the small domain of the Acy1/M20 family of enzymes was investigated.
  • the respective His269 from the lid domain of L delbrueckii PepV functions in the stabilization of the transition state. This residue corresponds to His229, His223 and His206 in the small domains of the dimeric homologs CPG2, PepT and hAcyl , respectively (Fig. 4C).
  • the two enzyme domains appear to be connected by flexible linkers, and show an open conformation.
  • the histidine is solvent exposed, and located 50 A from the metal center in the same monomer, and more than 8 A from the metal center in the opposite monomer of the dimer.
  • His229 in CPG2 coordinates an additional inter-dimeric zinc ion, which is further coordinated by Asp387 from another dimer within the crystallographic tetramer.
  • His223 in PepT contributes to the binding of a putative sulfate ion in the crystal structure, which was suggested to occupy a binding site for the C-terminal carboxyl group of the tripeptide substrate.
  • His206 in hAcyl was selected for mutation.
  • a His-to-Asn mutation generated an enzyme with a 2000-fold decrease in / ca t, and a by 10 °C reduced T m value (Table 2).
  • the complementation assay also showed that inactivation of the E147D mutant was not associated with irreversible unfolding of this protein.
  • the active site stabilities in the three combinations follow the order of the ⁇ G(H 2 O) values for the three mutants E147D, E147Q and D113A alone (Table 2). This likely reflects the impact of each of these mutations on the overall stability of the respective heterodimer with the H206N mutant.
  • the M28 family enzymes AAP and SGAP are believed to follow a zinc peptidase mechanism, which requires both metal ions for full activity. This mechanism is believed to be operational in all enzymes from the MH clan, which are, therefore, called co-catalytic metallopeptidases.
  • the dinuclear zinc center, and the putative general base residue, Glu147 in hAcyl, and in six available structures from the MH clan superimpose well (Fig. 2).
  • the results from the mutational analysis of hAcyl from the Acy1/M20 family corroborate the catalytic significance of a fully intact di-zinc center and Glu147.
  • an aspartate as zinc 2 ligand appears to provide the ability of family members to anchor a terminal amino group in their S1- pocket, thus determining aminopeptidase or dipeptidase over aminoacylase or carboxypeptidase specificities, which likely require a glutamate at this position. Accordingly, the disclosure herein helps to predict specificity from sequence. For instance, it appears that an aspartate at position 196 in DriP-1 indicates aminopeptidase or dipeptidase specificity for this enzyme. In the M28 family on the contrary, no similar correlation exists between zinc 2 ligation and substrate specificity as judged by the sequence alignment at the MEROPS database (http://merops.sanger.ac.uk).
  • an aspartic acid serves as a zinc 2 ligand, for instance, in the two genuine aminopeptidases AAP and SGAP, and also in human glutamate carboxypeptidase II, also known as prostate-specific membrane antigen.
  • the invention teaches the importance for enzyme activity of the conserved
  • Tyr384 and Tyr378 respectively, they exhibit very different side chain conformations, and could not be superimposed with Tyr246.
  • the conserved histidine in the small domain of Acy1/M20 family enzymes may play a similar role as Tyr246 in SGAP.
  • the results from the complementation assay (Fig. 4) indicate that in dimeric hAcyl this histidine is acting in trans.
  • a conformational change possibly induced by substrate-binding, brings two residues in the dimerization domain, a conserved histidine (His206 in hAcyl) and an asparagine (Asn263 in hAcyl), in the vicinity of the zinc center from the opposite monomer.
  • a highly conserved arginine (Arg276 in hAcyl) from the dimerization domain in the same monomer as the zinc center may be inserted in the active site.
  • the scheme in Figure 7 illustrates a corresponding model of the active site in hAcyl . In the following, the inventors tested the model shown in Figure 7 using mutants of the amino acid residues mentioned above.
  • Figure 8 shows that enzymatic activity is only recovered in the trans- combinations, arguing in favor of the predicted origin of active site residues. Remarkably, the inactivation of the N263D mutant was not associated with irreversible unfolding of this protein. However, the overall low recovery rates between 0.35 and 2.6 % of wild-type activity, with 16.7 % as the theoretical maximum, suggest impaired dimerization efficacy. The inventors demonstrated before the possible destabilizing impact of the mutations on the overall stability in
  • the conserved His229 residue from the dimerization domain of the one molecule in the GPG2 dimer contributes to the oxyanion hole together with the Zn1 ion of the other molecule. This may explain the importance of the corresponding His206 of hAcyl for catalysis as determined for three different mutations (Table 2 and 3).
  • the Asn275 residue of CPG2 is modeled in the proximity of, but not hydrogen-bonded to the C-terminal carboxylate of the substrate.
  • mutation of the hAcyl residue Asn263 to aspartic acid had a dramatic effect on the catalytic activity, which is not fully explained by the CPG2 model.
  • the present data also show that the alanine mutation of Asp274 in hAcyl reduces significantly the catalytic efficiency.
  • the corresponding Asp286 in the CPG2 model serves to anchor, and thus to correctly position the carboxylate-docking Arg288, without interacting directly with the substrate. This agrees with the almost unaltered K M value upon the D274A mutation of hAcyl (Table 3).
  • the catalytic base Glu174 forms two hydrogen bonds with the substrate in the transition state, one with the amide NH of the leaving group and the other one with the OH of the tetrahedral diol (formerly the hydroxylate nucleophile) ( Figure 10).
  • This modeled geometry which was not observed in the PepV-inhibitor complex (that lacks the scissile amide bond) strongly implicates the corresponding Glu147 of hAcyl as the general base in the hydrolysis.
  • the CPG2-substrate model also highlights other intermolecular interactions.
  • the two oxygen atoms of the tetrahedral diol of the transition state each coordinate a Zn-ion, and these interactions are likely to be preserved in the Acy1- substrate complex.
  • the acyl-binding S1 subsite is significantly hydrophobic.
  • the S1' subsite which accommodates the glutamate side chain in our model, extends in the opposite direction and is shaped by residues from both the Zn-binding and dimerization domains.
  • Low conservation between CPG2 and Acyl in both S1 and S1' regions are likely to account for their P1 and P1' selectivities, e.g., P1' preference for the glutamate or methionine side chains, respectively.
  • Wild-type hAcyl hydrolyzes N ⁇ -acylated amino acids with a preference for short acyl moieties (e.g. acetyl or formyl) in the Pi position. More bulky acyl moieties such as benzoyl-derivatives in this position, that is the S1 binding pocket, are not generally excluded but result in 100 to 1000-fold reduced catalytic efficiency due to lower /c ca t values (Fig. 3A). This renders the wild-type enzyme inactive against substrates of CPG2 such as the above-mentioned prodrugs.
  • short acyl moieties e.g. acetyl or formyl
  • More bulky acyl moieties such as benzoyl-derivatives in this position, that is the S1 binding pocket, are not generally excluded but result in 100 to 1000-fold reduced catalytic efficiency due to lower /c ca t values (Fig. 3A). This renders the wild-type enzyme inactive against substrates of
  • the concept of increasing the space or altering the shape of the S1 subsite by tageting amino acid positions 177, 187, 211 and 347 in hAcyl is an appropriate strategy to improve catalytic efficacy in the desired direction, i.e. against prodrug-like molecules resembling CPG2- or CPA-activated prodrugs.
  • CPG2 from Pseudomonas sp. is known to efficiently activate a range of tailor-made prodrugs.
  • the strong immunogenicity of CPG2 in humans severely limits the application of the enzyme in ADEPT.
  • the CPG2-nitrogen mustard prodrug systems are also considered for GDEPT.
  • Prodrugs which are excluded from cells should not be accessible to endogenous enzymes of strictly intracellular localization and, consequently, be safe from activation by such enzymes. This is indeed the case with nitrogen mustard prodrugs such as those activated by CPG2 or CPA.
  • the endogenous enzyme might be expressed only in tissues where it does not come in contact with the prodrug at any time after its administration.
  • a human enzyme could be engineered by means of chemical and/ or genetic modification in order to change its specificity toward the prodrug. Accordingly, and as the second aspect of the invention, determinants of substrate specificity in the active site of hAcyl were identified which is instrumental for such an engineering effort.
  • Wild-type and mutant hAcyl were expressed in a baculovirus expression vector system and purified as described (Pittelkow et al., 1998). In brief, 300 ml cultures of infected Sf21 cells were harvested 72 hours after infection. A two-step purification protocol was applied in all cases. The enzyme preparations were stored at -80 °C. Before use, protein samples were transferred into the appropriate buffer using a PD-10 column (Pharmacia). Protein was concentrated using centricon-10-concentrators (Amicon) following the instructions of the supplier. Protein concentrations were determined by the Bradford assay (Bradford, 1976) with BSA as the standard.
  • T m the 'melting temperature' T m

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Abstract

L'invention se rapporte à de l'aminoacylase d'enzyme cytosolique, et notamment sur ses variants, et sur des acides nucléiques les codant. L'invention se rapporte aussi à l'utilisation de ces variants et des acides nucléiques dans la thérapie à promédicament enzymatique dirigée (DEPT), telle que la thérapie à promédicament enzymatique dirigée anticorps (ADEPT) ou la thérapie à promédicament enzymatique dirigée gènes (GDEPT).
PCT/CA2004/000938 2003-06-23 2004-06-23 Variants d'aminoacylase, acides nucleiques les codant, et utilisations associees Ceased WO2004113524A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007136303A1 (fr) 2006-05-23 2007-11-29 Closed Company "Molecular-Medicine Technologies" Procédé d'identification d'une infection uro-génitale, oligonucléotide, combinaison d'oligonucléotides, biopuce et ensemble sur cette base
CN111699252A (zh) * 2019-01-16 2020-09-22 福尼亚生物处理股份有限公司 内切葡聚糖酶组合物和方法
CN115232804A (zh) * 2021-04-25 2022-10-25 上海医药工业研究院 一种重组羧肽酶g2突变体及其基因、制备方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077164A2 (fr) * 2001-03-23 2002-10-03 Pe Corporation (Ny) Aminoacylase humaine isolee, molecules d'acide nucleique codant l'aminoacylase humaine, et leurs utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077164A2 (fr) * 2001-03-23 2002-10-03 Pe Corporation (Ny) Aminoacylase humaine isolee, molecules d'acide nucleique codant l'aminoacylase humaine, et leurs utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LINDNER HOLGER A ET AL: "Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 45, 7 November 2003 (2003-11-07), pages 44496 - 44504, XP002300974, ISSN: 0021-9258 *
MITTA M ET AL: "The nucleotide sequence of human aminoacylase-1.", BIOCHIMICA ET BIOPHYSICA ACTA. 19 AUG 1993, vol. 1174, no. 2, 19 August 1993 (1993-08-19), pages 201 - 203, XP002300973, ISSN: 0006-3002 *
SPENCER D I R ET AL: "A STRATEGY FOR MAPPING AND NEUTRALIZING CONFORMATIONAL IMMUNOGENIC SITES ON PROTEIN THERAPEUTICS", PROTEOMICS, XX, XX, vol. 2, no. 3, March 2002 (2002-03-01), pages 271 - 279, XP009009265 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007136303A1 (fr) 2006-05-23 2007-11-29 Closed Company "Molecular-Medicine Technologies" Procédé d'identification d'une infection uro-génitale, oligonucléotide, combinaison d'oligonucléotides, biopuce et ensemble sur cette base
CN111699252A (zh) * 2019-01-16 2020-09-22 福尼亚生物处理股份有限公司 内切葡聚糖酶组合物和方法
CN111699252B (zh) * 2019-01-16 2024-04-30 福尼亚生物处理股份有限公司 内切葡聚糖酶组合物和方法
CN115232804A (zh) * 2021-04-25 2022-10-25 上海医药工业研究院 一种重组羧肽酶g2突变体及其基因、制备方法和应用
CN115232804B (zh) * 2021-04-25 2023-11-07 上海医药工业研究院 一种重组羧肽酶g2突变体及其基因、制备方法和应用

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