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WO2002067860A2 - Selection par dispositif d'affichage miroir - Google Patents

Selection par dispositif d'affichage miroir Download PDF

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Publication number
WO2002067860A2
WO2002067860A2 PCT/US2002/005193 US0205193W WO02067860A2 WO 2002067860 A2 WO2002067860 A2 WO 2002067860A2 US 0205193 W US0205193 W US 0205193W WO 02067860 A2 WO02067860 A2 WO 02067860A2
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carbohydrate
enantiomer
respect
peptide
binding activity
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PCT/US2002/005193
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WO2002067860A3 (fr
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Chi-Huey Wong
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The Scripps Research Institute
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Priority to AU2002250155A priority Critical patent/AU2002250155A1/en
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Publication of WO2002067860A3 publication Critical patent/WO2002067860A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the invention related to processes for selecting, receptors for cellular surface carbohydrates. More particularly, the invention relates to solid supported mirror-image sugars and to their use for selecting receptors of biologically important carbohydrates.
  • Carbohydrates on cell surfaces often form unique complex structures that act as key elements in various molecular recognition processes. Compounds that are capable of interfering with recognition of cell-surface carbohydrates thus hold great potential in biomedical applications. Such molecules, including inhibitors of carbohydrate-processing and synthesizing enzymes, cell-surface carbohydrate mimetics, as well as carbohydrate-binding antibodies, may be used as new drug candidates for the treatment of inflammation, cancer metastasis, bacterial or viral infection and as biosensors for detecting carbohydrates.
  • What is needed is effective means for identifying compounds that are capable of interfering with recognition of cell-surface carbohydrates, particularly compounds that are resistant to clearance mechanisms.
  • the invention is directed to processes and reagents employable for selecting D-peptides and/or L-nucleic acids (RNA or DNA) having binding activity with respect to naturally occurring sugars or carbohydrates.
  • the resultant D- peptide and/or L-nucleic acid binders are resistant to enzymatic degradation due to their non-naturally occurring chirality and are useful for blocking or activating the biological function of the naturally occurring sugar or carbohydrate to which they are targeted.
  • Preferred naturally occurring sugars and carbohydrates are involved in disease processes. Examples include bacterial or viral cell-surface sugars or carbohydrates, heparan sulfates involved in viral entry, thrombosis, and angiogenesis.
  • the selection process employs the enantiomer, i.e., the mirror image compound, of the targeted naturally occurring sugar or carbohydrate.
  • the enantiomer is synthesized or otherwise obtained and is then immobilized by attachment onto a support.
  • the immobilized enantiomer is then employed to screen libraries of L-peptides and/or D-nucleic acids to identify active binders.
  • Preferred libraries are phage display libraries.
  • binders may be mutagenized or otherwise modified and rescreened with respect to the immobilized enantiomer so as to obtain and identify binders having enhanced binding activity. This process of enhancement may be reiterated as desired or until no further enhancement is achieved.
  • the resultant L-peptides and/or D-nucleic acids having binding activity with respect to the target enantiomer are then isolated and identified.
  • Enantiomers of the identified L- peptides and/or D-nucleic acids i.e., D-peptides and/or L-nucleic acids corresponding to the identified L-peptides and/or D-nucleic acids, are then synthesized and tested for binding activity to the target naturally occurring sugar or carbohydrate.
  • the synthetic D-peptides and/or L-nucleic acids that bind to the target naturally occurring sugar or carbohydrate may then be employed for blocking or enhancing the biological activity of these targets.
  • One aspect of the invention is directed to a process for selecting an L- peptide binder that binds to an enantiomer of a naturally occurring sugar or carbohydrate.
  • the enantiomer of the naturally occurring sugar or carbohydrate is provided in a form employable for screening a library.
  • the library is screened against the enantiomer for identifying the L-peptide binder having binding activity with respect to the enantiomer.
  • the process may be extended by comparing the binding activity of the L-peptide binder both with respect to the enantiomer and with respect to the naturally occurring sugar or carbohydrate for determining the chiral specificity of the L-peptide binder identified above.
  • the process may be further extended synthesizing the enantiomer of the L-peptide binder identified above for obtaining a D-peptide having binding activity with respect to the naturally occurring sugar or carbohydrate. And finally, the process may be further extended by contacting the naturally occurring sugar or carbohydrate with the D-peptide, under binding conditions, for blocking or enhancing the biological activity of the naturally occurring sugar or carbohydrate.
  • Another aspect of the invention is directed to a process for obtaining a D- peptide having binding activity with respect to a first carbohydrate. The process employs first step of providing a second carbohydrate, the second carbohydrate being an enantiomer of the first carbohydrate, the second carbohydrate being employable for screening a library.
  • the library is screened against the second carbohydrate for identifying an L-peptide having binding activity with respect to the second carbohydrate.
  • the D-peptide which corresponding to the L-peptide is provided, the D-peptide having binding activity with respect to the first carbohydrate.
  • this process may be extended for binding a D-peptide to a first carbohydrate.
  • the D-peptide is contacted with the first carbohydrate under binding conditions for binding the D-peptide to the first carbohydrate.
  • Another aspect of the invention is directed to a process for selecting an D- nucleic acid binder that binds to an enantiomer of a naturally occurring sugar or carbohydrate.
  • the enantiomer of the naturally occurring sugar or carbohydrate is provided in a form employable for screening a library.
  • the library is screened against the enantiomer for identifying the D-nucleic acid binder having binding activity with respect to the enantiomer.
  • the process may be extended by comparing the binding activity of the D-nucleic acid binder both with respect to the enantiomer and with respect to the naturally occurring sugar or carbohydrate for determining the chiral specificity of the D-nucleic acid binder identified above.
  • the process may be further extended synthesizing the enantiomer of the D-nucleic acid binder identified above for obtaining a L-nucleic acid having binding activity with respect to the naturally occurring sugar or carbohydrate.
  • the process may be further extended by contacting the naturally occurring sugar or carbohydrate with the L- nucleic acid, under binding conditions, for blocking or enhancing the biological activity of the naturally occurring sugar or carbohydrate.
  • Another aspect of the invention is directed to a process for obtaining a L- DNA having binding activity with respect to a first carbohydrate.
  • the process employs first step of providing a second carbohydrate, the second carbohydrate being an enantiomer of the first carbohydrate, the second carbohydrate being employable for screening a library. Then, the library is screened against the second carbohydrate for identifying an D-DNA having binding activity with respect to the second carbohydrate. Then, the L-DNA which corresponding to the D-DNA is provided, the L-DNA having binding activity with respect to the first carbohydrate. In a preferred mode, this process may be extended for binding a L- DNA to a first carbohydrate. In this mode, the L-DNA is contacted with the first carbohydrate under binding conditions for binding the L-DNA to the first carbohydrate.
  • Another aspect of the invention is directed to a process for obtaining a L- RNA having binding activity with respect to a first carbohydrate.
  • the process employs first step of providing a second carbohydrate, the second carbohydrate being an enantiomer of the first carbohydrate, the second carbohydrate being employable for screening a library. Then, the library is screened against the second carbohydrate for identifying an D-RNA having binding activity with respect to the second carbohydrate. Then, the L-RNA which corresponding to the D-RNA is provided, the L-RNA having binding activity with respect to the first carbohydrate. In a preferred mode, this process may be extended for binding a L- RNA to a first carbohydrate. In this mode, the L-RNA is contacted with the first carbohydrate under binding conditions for binding the L-RNA to the first carbohydrate.
  • Another aspect of the invention is directed to an immobilized enantiomer of a naturally occurring sugar or carbohydrate employable for use in a selection process.
  • the carbohydrate product is an enantiomer of a first carbohydrate.
  • the first carbohydrate is of a type capable of being synthesized by a first enantiomeric aldol reaction when catalyzed by a first A/-acetylneuraminic acid aldolase having a first enantiomeric specificity for catalyzing the first enantiomeric aldol reaction.
  • the process includes the first step of converting the first ⁇ /-acetylneuraminic acid aldolase to a second ⁇ /-acetylneuraminic acid aldolase by directed evolution.
  • the second ⁇ /-acetylneuraminic acid aldolase has a second enantiomeric specificity for catalyzing a second enantiomeric aldol reaction.
  • the second enantiomeric aldol reaction is employable for synthesizing the carbohydrate product. Then, the carbohydrate product is synthesized by catalyzing the second enantiomeric aldol reaction with the second N- acetylneuraminic acid aldolase.
  • Figure 1 illustrates the design of D-peptides and L-nucleic acids that target cell-surface sugars.
  • Figure 2 illustrates the synthesis of L-NeuNAc (1 ) and the disaccharide L- NeuNAc-a-(2-3)-L-Gal (2).
  • Figure 3 illustrates a scheme showing the synthesis of aminoethylthiopropyl-L-sialic acid (14) from L-NeuNAc (1 ) in five steps.
  • Figure 4 illustrates three separate reactions or schemes.
  • Reaction a) shows the immobilization of aminoethylthiopropyl-L-sialic acid on different solid supports.
  • Reaction b) shows the conjugation of aminoethylthiopropyl-L-sialic acid to BSA.
  • Reaction c) shows the immobilization of aminoethylthiopropyl-L-KDO on different solid supports.
  • Figure 5 illustrates the schematic representation of the phage display method to select for antibodies against sugar-BSA conjugates.
  • FIG. 6 illustrates a table showing the protein sequences for selected single-chain human antibodies (scFv).
  • Figure 7 illustrates a table showing the dissociation constants (nM) for single chain human antibodies (scFv) K18, S11 , and S18.
  • Figure 8 illustrates the polyvalent interactions between carbohydrate conjugates and surface immobilized single-chain antibodies.
  • Figure 9 illustrates the synthesis of L-sialic acid-BSA conjugate.
  • Figure 10 illustrates the cycle used in the directed evolution of N- acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions.
  • Figure 11 illustrates the biopanning procedure for peptide binders using phage display.
  • the synthesis of the mirror image (the L-form) of the naturally occurring D-configurated carbohydrates is first obtained.
  • This L-sugar is then used to screen phage expressing a peptide library on the coat proteins to identify specific clones that bind to the L-sugar.
  • the mirror image of the L-peptide identified i.e. the corresponding D-peptide
  • the mirror image of the L-peptide identified is then chemically synthesized and the D-peptide should bind to the natural form of the target (i.e. naturally occurring D-sugar).
  • unnatural L-DNA can be created to target specific cell-surface carbohydrates. This approach provides a new technology platform to study sugar-protein and sugar-nucleic acid interaction.
  • L-NeuNAc 1 may be synthesized by either the neuraminic acid aldolase catalyzed addition of pyruvate to ⁇ /-acetyl-L-mannosamine (L-ManNAc) 5
  • L-ManNAc 5 was prepared from L-glucose as shown in Figure 2a.
  • L-NeuNAc 1 Enzymatic synthesis of L-NeuNAc 1 via the neuraminic acid aldolase catalyzed condensation of mannosamine 5 with pyruvic acid (Lin, C. H.; et al. J. Am. Chem. Soc. 1992, 114, 10138-10145) was too slow to be useful for preparative synthesis.
  • L-NeuNAc 1 was synthesized in 28 % yield by chemical condensation of L-ManNAc 5 and the potassium salt of di-f-butyl oxalacetate 11 (Figure 2b). (Kuhn, R.; Baschang, G. Justus Liebigs Ann. Chem. 1962, 659, 156-161 ) Compound 1 was further linked to L-galactose to provide 2.
  • D-NeuNAc-( ⁇ -2,3)-D-Gal exists as a common moiety in typical tumor-associated glycosphingolipid antigens and the binding site of influenza virus hemagglutinin (Hakomori, S.; Zhang, Y. Chem.& Biol. 1997, 4, 97-104; Ito, Y.; et al. Pure Appl. Chem. 1993, 65, 753-762).
  • the enantiomer 2 was synthesized by coupling of L-sialyl phosphite 9 with 6-TBDPS-1-O-allyl-L-galactose 10, which was prepared from L-galactose through 1-O-allyl-2,3,4,6-tetraacetate-L-galactose 12 as shown in Figure 2c.
  • the L-sialyl phosphite 9 was synthesized from L-sialic acid 1 by methylation of the carboxylic acid, acetylation of all the free hydroxyl groups, exchange the anomeric acetate to phosphite.
  • D-KDO and D-KDN are key components of the core region of bacterial lipopolysaccharides (Hansson, J.; Oscarson, S. Curr. Org. Chem. 2000, 4, 535- 564).
  • the enantiomers L-KDO 3 and L-KDN 4 were prepared on gram scales by neuraminic acid aldolase-catalyzed addition of pyruvate to L-arabinose or L-mannose, respectively, according to modified literature procedures (Gautheronlenarvor, C; et al. J. Am. Chem. Soc. 1991 , 113, 7816-7818; Lin, C. H.; et al. J. Am. Chem. Soc. 1992, 114, 10138-10145). We found that an increased amount of the enzyme was necessary to achieve high yield.
  • 3-(2-aminoethyl-thio)-propyl moiety was chosen as the linker. It can be readily synthesized by irradiating allyl glycosides in the presence of cysteamine.
  • Aminoethylthiopropyl-L-sialic acid 14 was prepared in five steps from L-NeuNAc 1 ( Figure 3) (Roy, R.; Laferriere, C. A. Carbohydr. Res. 1988, 177, d-c4; Roy, R.; Laferriere, C. A. Can. J. Chem. 1990, 68, 2045-2054; Laferriere, C. A.; et al. Meth.
  • the aminoethylthiopropyl L-NeuNAc 14 and L-KDO 17 were immobilized through an amide bond on a number of solid supports such as Affi-Gel 15, Affi-Prep 10, Dynabeads M-270, and BIACore sensor chip CM5 ( Figure 4a).
  • the library was subjected to four rounds of panning using Maxisorb Nunc plates coated with either D-KDO-BSA or D-NeuNAc-BSA (Figure 5). Phage were pooled after each successive round of panning and tested for their ability to bind either BSA conjugate. The enrichment clone number using either conjugate went from 2 x 10 5 to 8 x 10 8 . Thus, after the fourth round of panning a total of 40 phage clones (20 to D-KDO-BSA and 20 D-NeuNAc-BSA) were randomly selected that survived the rigorous individual selection process. It was found that 14 and 16 clones exhibited substantial binding activity to
  • D-KDO-BSA and D-NeuNAc-BSA were collected from the unpanned library, and none of these clones showed any affinity (as determined by ELISA) to either D-KDO-BSA or D-NeuNAc-BSA.
  • Clones with the greatest affinity as judged by ELISA were subjected to sequencing, and the nucleotide sequences encoding the V H and V L were determined.
  • D-NeuNAc clones picked two sequences were found that differed in the V H and V L regions, while of the 7 D-KDO clones chosen a single consensus sequence for both the V H and V L chains was uncovered ( Figure 6).
  • Figure 7 illustrates the specificity and affinity of the single-chain antibodies for the carbohydrate conjugates as determined by SPR. Since the antibodies were selected against D-sugars, they showed greater affinity for their respective D-sugar conjugates versus the L-sugar conjugates. The difference in dissociation constants is very significant between D-NeuNAc-BSA and L-NeuNAc-BSA. As expected, K18 displayed the greatest specificity for D-KDO-BSA as compared with L-KDO-BSA, since it was selected against D-KDO during the panning process. The antibodies selected against D-sugars, they showed greater affinity for their respective D-sugar conjugates versus the L-sugar conjugates. The difference in dissociation constants is very significant between D-NeuNAc-BSA and L-NeuNAc-BSA. As expected, K18 displayed the greatest specificity for D-KDO-BSA as compared with L-KDO-BSA, since it was selected against D-KDO during the panning process. The antibodies
  • D-NeuNAc, S11 and S18 displayed the greatest specificity for D-NeuNAc-BSA as opposed to L-NeuNAc-BSA, but showed no specificity between the D- and L-KDO-BSA.
  • As a control experiment no interaction between the antibodies and BSA that contains only conjugated linker but no sugar was observed.
  • the mechanism of binding of the carbohydrate conjugates to the immobilized antibodies appears to be by polyvalent interactions (Figure 8). Unconjugated D-KDO (up to 50 mM) showed no interactions with any of the antibodies, and unconjugated D-NeuNAc showed binding at concentrations of 12.5 mM and above as judged by SPR (data not shown).
  • NaHepes/150mM NaCI pH 7.4 to remove any residual BSA.
  • the prepared plates were stored in 50mM NaHepes/150mM NaCI (pH 7.4) at 4 °C.
  • Ten wells were prepared for five rounds of panning for each set of experiments, five with sugar conjugated BSA (for the binding experiment) and five with only BSA (for the control experiment).
  • the wells were washed twice with 180 ⁇ L of 50mM NaHepes (pH 7.4).
  • 20 ⁇ L of the stock 12mer Phage Display Library (New England BioLabs) was diluted with 180 ⁇ L of 50mM NaHepes (pH 7.4).
  • 100 ⁇ L of the phage solution was added to the well containing sugar conjuaged BSA.
  • the other 100 ⁇ L was added to the well containing only BSA. Incubation was allowed to occur overnight at 4 °C.
  • an overnight culture of 10mL of ER2738 E. coli (New England BioLab) was prepared in LB media and 15 ⁇ g/mL tetracycline for the phage amplification step next day.
  • the phage solution from each well was pipetted off and discarded. Each well was washed twice with 180 ⁇ L of 50mM NaHepes/0.1% Tween (pH 7.4) to remove background binders. The bound phage was eluted by incubating 100 ⁇ L of 5mM L-Sialic Acid in 50mM NaHepes (pH 7.4) for 2 hrs at 4 °C. 1 ⁇ L of eluted phage was used for titering (see below) while the rest was amplified in the next step. Phage Amplification and Isolation
  • the phage was harvested by centrifugation at 2,000 rpm in 50 mL culture tubes at 4 °C.
  • the amplified phage was resuspended in 400 ⁇ L of 50mM NaHepes/150mM NaCI (pH 7.4) and stored at 4 °C. The first round of panning was accomplished.
  • the amplified phage was diluted with 50mM Na Hepes (pH 7.4) and the panning process begins again starting with the Phage and Ligand Incubation step.
  • background phage binders was removed with 10 wash cycles with 50mM NaHepes (pH 7.4)/0.1 % Tween. Five rounds of panning was done to isolate the L-Sialic Acid binder.
  • the overnight culture of ER2738 was diluted 1 :100 in LB media and dispensed in 2mL volumes into 15mL sterile culture tubes. With a sterile wooden stick, a stab of blue/green plaque was transferred to a culture tube containing the diluted cells. 10 plaques from the 5th round were picked and grown for 5 hrs at 37 °C. 1.2mL of the each culture was transferred to the microcentrifuge tube and centrifuged for 10 minutes. 1 mL of the phage containing supernatant was then transferred to a fresh microcentrifuge tube and 400 ⁇ L of 20% PEG-8000/2.5M NaCI was added to it. The solution was mixed and let stand at room temperature for 10 minutes.
  • the precipitated phage was then harvested by centrifugation for 10 minutes and the supernatant was discarded. The tube was centrifuged again and the remaining trace supernatant was pipetted off. The phage pellet was suspended in 100 ⁇ L Iodide Buffer (1 OmM Tris-HCI/1 mM EDTA/4M Nal pH 8.0) and 250 ⁇ L of ethanol was added to the tube. The solution was incubated for 10 minutes at room temperature and the single-stranded phage DNA was harvested by centrifugation for 10 minutes. The DNA pellet was washed with 70% ethanol to remove the remaining salts and dried under vacuum. The pellet was resuspened in distilled water and the DNA was sequenced using automated DNA sequencing methods.
  • Iodide Buffer 1 OmM Tris-HCI/1 mM EDTA/4M Nal pH 8.0
  • the first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the ⁇ / ⁇ -barrel active site exihibits an inversion of enantioselectivity toward KDO
  • the second generation variant contains additional amino acid changes (Val251 lie) outside the ⁇ / ⁇ -barrel active site that significantly improves the enantiomeric reactions for L-sialic acid and L-KDO.
  • ⁇ /-acetylneuraminic acid aldolase catalyses the reversible aldol reaction of ⁇ /-acetyl-D-mannosamine and pyruvate to give ⁇ /-acetyl- D-neuranominic acid (D-sialic acid) and has been extensively used in the synthesis of sialic acids and analogs (Kim, M. J.; et al. J. Am. Chem. Soc. 1988, 110, 6481-6486; Lin, C.-C; Lin, C.-H.; Wong, C.-H. Tetrahedron Lett. 1997, 38, 2649-2652; Kragl, U.; et al. J. Chem.
  • Neu ⁇ Ac aldolase has been shown to be specific for pyruvate as the donor, but flexible to a variety of hexoses and pentoses, both D- and to some extent L- sugars, as acceptor substrate (Lin, C. H.; et al. J. Am. Chem. Soc 1992, 114, 10138-10145; Gautheron-Le Narvor, C; et al. J. Am. Chem. Soc. 1991 , 113, 7816-7818). It is disclosed herein that the enantioselectivity of this enzyme has been engineered to develop an effective catalyst for the synthesis of both D- and L-sugars.
  • Assay samples containing D- or L-KDO, the evolved crude enzyme, and the coupled system were prepared on 96-well plates and absorbance of NADH at 340 nm was monitored over time with a micro-titer plate reader. Screening of enzymes with low initial activities could be facilitated by the presence of a sufficient amount of enzyme in each sample, such that a good signal to noise ratio could be obtained over a reasonable period of assay time.
  • the N-terminal 6x histidine fusion expression vector, pTrcHisB (Invitrogen) was chosen for our studies of Neu ⁇ Ac aldolase as it provides a high expression system and the availability of rapid single step affinity purification of the expressed protein for further analysis. Subsequent libraries of mutant Neu ⁇ Ac aldolase genes were cloned into this vector.
  • the wild-type aldolase gene was amplified under standard mutagenic PCR condition with a controlled mutation rate of 1-5 bases per gene to generate the first generation plasmid library.
  • the protein library was prepared by lysis of re-suspended cell pellets harvested from 2mL cultures of individually picked colonies obtained by transformation with the plasmid library. The lysed suspensions were then heat treated to reduce background interference due to other contaminants present in the cell lysate, and the supernatants were transferred to 96-well plates for assays at 25 °C. About 25% of the first generation mutants still retained certain activity after the heat treatment. Hence, we adopted this strategy for reducing the background signal and for a more stringent evolution condition that restrains the drifting of thermal stability of selected mutants.
  • Mut#1 , Mut#2, and Mut#3 had the same mutations.
  • DNA shuffling was done to produce the second generation library using Mut#1 and Mut#4. Eight 0 hundred variants of this library were screened for both D- and L-KDO cleavage; however, no mutant with improved activity toward L-KDO was identified. Thus, the best first generation mutant (Mut#1 ) was selected as a template for another round of mutagenic PCR and screening. 1 ,600 variants of the error-prone PCR products were screened and one mutant (designated as Mut#2-5) with 5 cleavage activity toward L-KDO (0.06% screened population) was identified for further characterization. Characterization of the catalytic properties of the evolved Neu ⁇ Ac aldolases
  • the selected mutants were purified and analyzed for their ability to cleave D- and L- KDO.
  • Both the first generation mutant Mut#1 and the second 0 generation mutant Mut#2-5 have higher k ⁇ and lower K m toward D-KDO compared to the wild-type.
  • Their specificity constant (k ⁇ t/ K m ) toward D-KDO is 3.3- and 3.6-fold increase compared to the wild-type, respectively. Progressive improvement of both / cat and K m was observed in the evolution of the aldolase toward L-KDO cleavage.
  • the second generation ⁇ mutant Mut#2- ⁇ has a 1.6-fold increase in k ⁇ and a 1. ⁇ -fold reduction in m for L-KDO and hence a 2.4-fold improvement in k ⁇ K m .
  • Mut#2- ⁇ has a 30-fold increase in k ⁇ toward L-sialic acid compared to the wild type, while the / cat toward D-sialic acid remains basically the same.
  • the K m value is much higher for L-sialic acid, resulting in a relatively low / cat , m value.
  • the substrate concentration is often relatively high so that the enzyme operates at the maximum velocity.
  • Mut#1 and Mut#2-5 exhibit higher k ⁇ toward D- and L-KDO compared to the wild type.
  • Mut#2-5 showed slightly better L-enantioselectivity toward KDO (0.27 for L vs. 0.25 for D) while no inversion of enantioselectivity was observed for KDO or sialic acid.
  • an inversion of enantioselectivity toward KDO was observed for Mut#1.
  • the wild-type enzyme has remarkable high substrate specificity for D-sialic acid over L-KDO
  • the evolved enzyme Mut#2-5 is significantly less specific for these substrates and an improved L-enantioselectivity was observed with KDO.
  • ⁇ /-acetyl-L-mannosamine to pyruvate.
  • ⁇ /-acetyl-L-mannosamine has been reported as a very weak substrate for the wild-type E. coli Neu ⁇ Ac aldolase (Kim, M. J.; et al. J. Am. Chem. Soc 1988, 110, 6481-6486) (at -0.6% the rate for ⁇ /-acetyl-D-mannos-amine), but no detectable activity under the current assay condition.
  • Mut#2- ⁇ exhibits a relatively high k ⁇ for L-sialic acid, a relatively high concentration of substrate is required, however, as the affinity for the sugar substrate is relatively high.
  • Mut#2- ⁇ for sialic acid and for N-acetyl-L-mannosamine.
  • the activities of Mut#2- ⁇ toward L-and D- arabinose are relatively high compared with wild-type and have been used in the preparative synthesis of D- and
  • D-KDO was synthesized by chemical condensation of D-arabinose and oxalacetate as described previously (Kozlov, I. A.; et al. ChemBioChem. 2001 , 2, 741-746).
  • L-KDO was made by enzymatic synthesis (Kim, M. J.; et al. J. Am. Chem. Soc 1988, 110, 6481-6486; Kozlov, I. A.; et al. ChemBioChem. 2001 , 2, 741-746) using the wild-type Neu ⁇ Ac aldolase expressed in E. coli.
  • Both enantiomers of KDO were purified by AG-1 X-8 (HCO 3 " ) anion-exchange resin (Bio-rad) and re-crystallization from ethanol-water to remove pyuruvate ⁇ contamination.
  • the 0.9-kb E. coli Neu ⁇ Ac aldolase gene was amplified by standard PCR from a genomic preparation of E. coli JM109, with primers N-NeuA 0 ( ⁇ '-ATC GCG GAT CCG ATG GCA ACG AAT TTA CGT G) (SEQ ID NO 33) and C-NeuA ( ⁇ '-ATC CGG AAT TCT CAC CCG CGC TCT TGC ATC) (SEQ ID NO 34) flanking the gene with restriction sites BamH I and EcoR I.
  • the resulting fragment was purified, doubly digested with BamH I and EcoR I and was ligated into vector pTrcHisB (Invitrogen) digested with the same restriction ⁇ enzymes.
  • the ligation product was transformed into E. coli XL1 Blue-MRF' by electroporation (Dower, W. J.; et al. Nucleic Acids Res. 1988, 16, 6127-614 ⁇ ). Plasmids recovered from transformants were screened by PCR for the presence of the aldolase insert. A positive plasmid clone ⁇ /eu>A-pTrcHisB was sequenced, used for protein expression and as mutation template for the 0 construction of the first generation library.
  • Mutagenic PCR was carried out under standard error-prone condition (Eckert, K. A.; Kunkel, T. A. Nucleic Acids Res. 1990, 18, 3739-3744). Primers N-NeuA and C-NeuA were used to amplify and mutate the template gene.
  • the library fragment was 0 gel-purified and cloned into pTrcHisB as described above and the resulting library construct was transformed into XL1 Blue-MRF', amplified and purified as plasmid miniprep (Qiagen).
  • the presence of gene insert in the library constructs was confirmed by gel-electrophoresis with the parent vector pTrcHisB as reference.
  • Heterogeneity of the first generation library construct was examined by transforming the plasmid into JM109 following by random picking of three colonies, plasmid extraction, and DNA sequencing. Each of the selected mutants contained one to five mutations. DNA shuffling was done according to the method of Stemmer (Stemmer,
  • Constructs harboring the mutant library were transformed into JM109 by electroporation.
  • the transformed culture was spread on Luria-Bertani (LB) agar plates containing 50 ⁇ g/mLof ampicillin and incubated at 37 °C for 16 hours. Individual colonies were picked, replicated on a LB agar-ampicilin plate, and dispensed into 24-well plates that contained 2 ml of 2X YT/50 ⁇ g ml "1 ampicillin/0.2 mM IPTG (the broth was shaken vigorously before dispensing into the plates).
  • LB Luria-Bertani
  • the plates were sealed and shaken at 37 °C/200 rpm in a shaker-incubator for 18 hours, centrifuged at 4000 rpm/4 °C for one hour, and the supernatant was carefully decanted. Each cell pellet was re-suspended in 0.4 ml of ⁇ O mM potassium phosphate buffer, pH 7. ⁇ containing O. ⁇ mg/mL of lysozyme. The plates were rapidly frozen in liquid nitrogen followed by thawing at room temperature, and then incubated at 65 °C for 20 minutes. Cell debris was collected by centrifugation at 4000 rpm, 4 °C for one hour.
  • Selected plasmids were transformed into JM109.
  • Cell pellet from 600 ml culture was 0 re-suspended in 20 ml of ⁇ O mM potassium phosphate pH 7. ⁇ / ⁇ mM ⁇ -mercaptoethanol/300 mM NaCI, chilled on ice, and was lysed by passing through a French Press (SLM instruments, Urbana, IL) compressed to 1 ,600 Psi and then released to ambient pressure. The process was repeated three times. Cell debris was pelleted by centrifugation at 12,000g, 4 °C for 1 hour.
  • SLM instruments Urbana, IL
  • the supernatant was filtered through a 0.2 micron cellular acetate membrane filter (Corning), and was loaded onto a Ni 2+ -NTA-agarose column with a bed volume of 2. ⁇ ml pre-equilibrated with the cell re-suspension buffer.
  • the column was washed with 20 ml of buffer containing ⁇ O mM potassium phosphate pH 7.5/ ⁇ mM ⁇ -mercaptoethanol/300 mM NaCI/ ⁇ % glycerol/10 mM 0 imidazole, and then 20 ml of buffer containing 60 mM potassium phosphate pH 7. ⁇ / ⁇ mM ⁇ -mercaptoethanol.
  • Bound enzyme was eluted with 60 mM potassium phosphate pH 7. ⁇ / ⁇ mM ⁇ -mercaptoethanol/2 ⁇ 0 mM imidazole, and was dialyzed extensively against ⁇ O mM potassium phosphate pH 7. ⁇ / ⁇ mM ⁇ -mercaptoethanol at 4 °C. Eluted enzymes were analyzed with SDS-PAGE and were found to be >90% pure in all cases. Enzyme solution was frozen in liquid nitrogen and was stored at -78 °C prior to use. No activity lost was observed upon freezing and thawing the enzymes. Enzyme concentrations ⁇ were determined by Bradford procedure (Bio-Rad) using BSA as calibration standard.
  • the activity was determined by the standard coupled assay with L-lactic 0 dehydrogenase (EC 1.1.1.27, Type II from rabbit muscle) and NADH.
  • a typical assay was initiated by addition of an appropriate amount of D-KDO or L-KDO in ⁇ O mM potassium phosphate, pH 7. ⁇ / ⁇ mM ⁇ -mercaptoethanol, to a mixture of L-lactic dehydrogenase (0.8 U)/NADH (0.43 mM)/ aldolase (10-100 ⁇ g) in 60 mM potassium phosphate, pH 7. ⁇ / ⁇ mM ⁇ -mercaptoethanol.
  • the total reaction ⁇ volume was 800 ⁇ l.
  • the mixture Prior to the addition of the substrate, the mixture was pre-incubated at 26 °C for ⁇ minutes. UV absorbance at 340 nm was recorded continuously for two minutes and the slope of the absorbance curve during the first 30 seconds was used for rate estimation.
  • the aldol condensation activity was determined by the rate of depletion of pyruvate. Pyruvate concentration was determined by a method similar to that previously reported (Gautheron-Le Narvor, C; et al. J. Am. Chem. Soc 1991 , 113, 7816-7818). Reactions were initiated by the addition of the 6 aldolase to a mixture of pyruvate and sugars preincubated at 37 °C. Reactions were performed in 1 ml of 60 mM phosphate buffer, pH 7.6. Starting concentrations of pyruvate, sugars and enzyme were 10 mM, 260 mM and 30-300 ⁇ g/mL, respectively.
  • Figure 1 shows the design of D-peptides and L-nucleic acids that target cell-surface sugars.
  • the enantiomer of a cell-surface sugar (L-sugar) is used as an affinity ligand for identification of L-peptides (from a phage display library) ⁇ or D-DNA or D-RNA (via in vitro evolution) that bind the ligand.
  • L-sugar L-sugar
  • D-DNA or D-RNA via in vitro evolution
  • Figure 2 shows the synthesis of L-NeuNAc (1) and the disaccharide L- 0 NeuNAc-a-(2 ⁇ 3)-L-Gal (2). Reagents and conditions: a) BnOH, TsOH; b)
  • PhCH(OMe) 2 camphor sulfonic acid, 66%; c) Tf 2 0, pyridine, CH 2 CI 2 ; d) NaN 3 , DMF, 72%; e) H 2 , Pd(OH) 2 /C, MeOH, Ac 2 0; f) PPh 3 , THF followed by Ac 2 0, MeOH, 88%; g) H 2 , Pd(OH) 2 /C, MeOH, AcOH, 100%; h)Me0H,NiCI 2 /H 2 0, 70%, 2h, 28% ; i) Ac 2 0, NaOAc, reflux, 3h, 48%; j) BF 3 « Et 2 0, allyl alcohol, CH 2 CI 2 , 0 ⁇ °C, 2h, 60%; k) NaOMe, MeOH, rt, 1 h; I) TBDPSCI, DMF, imidazole, rt, 2h,
  • FIG. 3 is a scheme showing the synthesis of aminoethylthiopropyl-L- sialic acid (14) from L-NeuNAc (1) in five steps.
  • Reagents and conditions a) MeOH, HCl, rt, 12h; b) AcCI, AcOH, MeOH, rt, 48h, 94%; c) allyl alcohol, silver(l) salicylate, 3 A molecular sieves, 2 ⁇ °C, 18 h; d) MeOH, NaOMe, rt, 1 h; e) NaOH, H 2 0, rt, 1 h, 60%; f) HSCH 2 CH 2 NH 2 , H 2 0, UV, 18 h, 7 ⁇ %.
  • Aminoethylthiopropyl-L-KDO (17) and aminoethylthiopropyl-L-KDN (18) were obtained in a similar manner.
  • Reaction a) shows the immobilization of aminoethylthiopropyl-L-sialic acid on different solid supports.
  • the aminoethylthiopropyl L-NeuNAc 14 and L-KDO 17 were 0 immobilized through an amide bond on a number of solid supports such as Affi-Gel 1 ⁇ , Affi-Prep 10, Dynabeads M-270, and BIACore sensor chip CM ⁇ .
  • Reaction b) shows the conjugation of aminoethylthiopropyl-L-sialic acid to BSA.
  • compounds 14 and 17 were immobilized on Nunc immunoplates with two approaches.
  • Figure 5 illustrates the schematic representation of the phage display method to select for antibodies against sugar-BSA conjugates.
  • the library was subjected to four rounds of panning using Maxisorb Nunc plates coated with either D-KDO-BSA or D-NeuNAc-BSA. Phage were pooled after each successive round of panning and tested for their ability to bind either BSA conjugate. The enrichment clone number using either conjugate went from 2 x 10 5 to 8 x 10 8 . Thus, after the fourth round of panning a total of 40 phage clones (20 to D-KDO-BSA and 20 D-NeuNAc-BSA) were randomly selected that survived the rigorous individual selection process. It was found that 14 and 16 clones exhibited substantial binding activity- to D-KDO-BSA and D-NeuNAc-BSA, respectively, as determined by ELISA.
  • Figure 6 is a table showing the protein sequences for selected single-chain human antibodies (scFv).
  • FR Framework region
  • CDR complementarity-determining region.
  • Clone K18 was selected from D-KDO
  • clones S11 and S18 were selected from D-NeuNAc.
  • Figure 7 is a table showing the dissociation constants (nM) for single chain human antibodies (scFv) K18, S11 , and S18.
  • the dissociation constants were measured by the method in which the single-chain antibodies were immobilized on the BIACORE CM5 chip surface and the carbohydrate-BSA conjugate was injected into the flow cell at different concentrations.
  • K18 was selected from D-KDO.
  • S11 and S18 were selected from D-NeuNAc.
  • Figure 8 illustrates the polyvalent interactions between carbohydrate conjugates and surface immobilized single-chain antibodies.
  • the interaction was detected by surface plasmon resonance on the CM ⁇ chip surface via binding experiments using BIACORE 2000.
  • Unconjugated D-KDO up to 50 mM
  • unconjugated D-NeuNAc showed binding at concentrations of 12.5 mM and above as judged by SPR (data not shown).
  • the carbohydrates were immobilized on the CM ⁇ chip and the single-chain antibodies were passed over the chip surface, no significant interactions were observed in the micromolar range. Because each carbohydrate conjugate consists of 12 sugars linked to BSA (see ⁇ Experimental Section), the observed nanomolar dissociation constants are probably a result of polyvalent interactions between the two interacting species.
  • Figure 9 shows the synthesis of L-sialic acid-BSA conjugate.
  • the scheme shows that 12 sialic acids are attached to one BSA protein.
  • Figure 10 shows the cycle used in the directed evolution of N- 0 acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions.
  • Figure 11 illustrates the biopanning procedure for peptide binders using phage display.
  • 20 ⁇ L of the stock 12mer Phage Display Library (New England BioLabs) was diluted with 180 ⁇ L of ⁇ OmM NaHepes (pH 7.4).
  • 100 ⁇ L of the phage solution was added to the 5 well containing sugar conjuaged BSA (Step l ).
  • the phage solution from each well was pipetted off and discarded.
  • Each well was washed twice with 180 ⁇ L of ⁇ OmM NaHepes/0.1 % Tween (pH 7.4) to remove background binders (Step 2).
  • the bound phage was eluted by incubating 100 ⁇ L of ⁇ mM L-Sialic Acid in ⁇ OmM NaHepes (pH 7.4) for 2 hrs at 4 °C (Step 3). 1 ⁇ L of eluted phage was 0 used for titering while the rest was amplified in the next step. The eluted phage was added to the cell culture and the cells were allowed to grow for ⁇ hrs at 37 °C to amplify the bound phage (Step 4). The cell culture was then centrifuged for O. ⁇ hr at 8,000 rpm to remove the cells.
  • the phage which resides in the supernatant, was precipitated with 20mL of 20% (w/v) PEG-8000/2.5M NaCI for 5 1 hr. at 4 °C.
  • the phage was harvested by centrifugation at 2,000 rpm in 50 mL culture tubes at 4 °C (Step 5).
  • the amplified phage was resuspended in 400 ⁇ L of 50mM NaHepes/150mM NaCI (pH 7.4) and stored at 4 °C. The first round of panning was accomplished.

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Abstract

Selon l'invention, on identifie les liants artificiels se trouvant dans les glucides et les sucres des surfaces cellulaires au moyen d'un procédé de criblage qui utilise les énantiomères immobilisés de ces glucides et sucres de surfaces cellulaires. Les liants artificiels préférés comprennent des D-peptides et des L-acides nucléiques et résistent à la dégradation enzymatique et à l'épuration. Par ailleurs, ce procédé à permis d'identifier des séquences Fab à chaîne simple qui se lient à l'acide sialique et au KDO selon une affinité nano-molaire. Des exemples de procédures de criblage utilisent des D-KDO, L-acide sialique et L-sialo-disaccharide fixés sur un support solide pour sélectionner des liants à forte affinité.
PCT/US2002/005193 2001-02-22 2002-02-22 Selection par dispositif d'affichage miroir WO2002067860A2 (fr)

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EP2045326A1 (fr) * 2007-10-05 2009-04-08 Bayer CropScience AG Procédé d'identification du partenaire d'interaction d'un agent actif

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WO1997035194A2 (fr) * 1996-03-21 1997-09-25 President And Fellows Of Harvard College Procede de criblage enantiomere et compositions pour ce procede
US5780221A (en) * 1995-05-03 1998-07-14 Whitehead Institute For Biomedical Research Identification of enantiomeric ligands
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US5869461A (en) * 1995-03-16 1999-02-09 Yale University Reducing toxicity of L-nucleosides with D-nucleosides
US5780221A (en) * 1995-05-03 1998-07-14 Whitehead Institute For Biomedical Research Identification of enantiomeric ligands
WO1997035194A2 (fr) * 1996-03-21 1997-09-25 President And Fellows Of Harvard College Procede de criblage enantiomere et compositions pour ce procede
US6114309A (en) * 1997-11-21 2000-09-05 Incara Research Laboratories Combinatorial library of moenomycin analogs and methods of producing same
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2045326A1 (fr) * 2007-10-05 2009-04-08 Bayer CropScience AG Procédé d'identification du partenaire d'interaction d'un agent actif
WO2009043565A1 (fr) * 2007-10-05 2009-04-09 Bayer Cropscience Ag Méthode d'identification du partenaire d'interaction d'un agent actif

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