WO2020168061A1 - Compositions et procédés de criblage de protéase - Google Patents

Compositions et procédés de criblage de protéase Download PDF

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Publication number
WO2020168061A1
WO2020168061A1 PCT/US2020/018089 US2020018089W WO2020168061A1 WO 2020168061 A1 WO2020168061 A1 WO 2020168061A1 US 2020018089 W US2020018089 W US 2020018089W WO 2020168061 A1 WO2020168061 A1 WO 2020168061A1
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Prior art keywords
chimeric
peptide substrate
content
substrate
protease
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PCT/US2020/018089
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English (en)
Inventor
Ward C. Tucker
Francis Mark DUNNING
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Biomadison, Inc.
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Publication of WO2020168061A1 publication Critical patent/WO2020168061A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the field of the invention is identification of protease activity, particularly from genetically modified proteases.
  • Enzymes in particular proteases, have found use as therapeutic agents.
  • Botulinum neurotoxins BoNTs
  • BoNTs Botulinum neurotoxins
  • BoNTs Botulinum neurotoxins
  • the resulting long-lasting decrease in motor neuron exocytotic activity has found wide therapeutic and cosmetic applications.
  • Therapeutic use of proteases has, however, been limited by the lack of naturally occurring proteases with specificity and/or activity that impacts target disease states, such as disease associated with hypersecretion.
  • inventive subject matter provides apparatus, systems and methods in which a—
  • FIG. 1 depicts a SNAP-25 based reporting construct and an exemplary set of chimeric reporting constructs that incorporate recombinant chimeric peptide substrates having different degrees of SNAP-23 content, which are useful for screening Botulinum neurotoxin serotype A (BoNT/A) light chain mutations for activity with SNAP-23.
  • BoNT/A Botulinum neurotoxin serotype A
  • FIG. 2 depicts alternative exemplary chimeric peptide substrates useful for screening Botulinum neurotoxin serotype A (BoNT/A) light chain mutations for activity with SNAP-23.
  • BoNT/A Botulinum neurotoxin serotype A
  • FIG. 3A depicts an example of a chimeric reporting construct that incorporates a chimeric peptide substrate of the inventive concept.
  • the chimeric peptide substrate includes a relatively small portion of SNAP-23 (14 amino acids) and amino acids 173- 206 of SNAP-25.
  • FIG. 3B depicts another example of a chimeric reporting construct that incorporates a chimeric peptide substrate of the inventive concept.
  • the chimeric peptide substrate includes a larger portion of SNAP-23 (36 amino acids) than that in the example in FIG. 3 A and amino acids 182-206 of SNAP-25.
  • FIG. 3A depicts an example of a chimeric reporting construct that incorporates a chimeric peptide substrate of the inventive concept.
  • the chimeric peptide substrate includes a relatively small portion of SNAP-23 (14 amino acids) and amino acids 173- 206 of SNAP-25.
  • FIG. 3B depicts another example of a chimeric reporting construct that incorporates
  • 3C depicts another example of a chimeric reporting construct that incorporates a chimeric peptide substrate of the inventive concept.
  • the cleavage site includes a larger portion of SNAP-23 (47 amino acids) than that in the example in FIG. 3B and amino acids 193-206 of SNAP-25.
  • FIG. 4 schematically depicts an exemplary evolutionary approach to modifying binding and/or substrate specificity of Botulinum neurotoxin serotype A light chain (BoNT/LC).
  • FIG. 5 schematically depicts an exemplary screening assay for binding to chimeric peptide substrates of the inventive concept.
  • FIG. 6A shows results of screening of BoNT LC/A mutations
  • FIG. 6B shows results of screening of BoNT LC/A mutations Mutl2 (E148) through Mut23 (E148). As shown, no mutations resulted in cleavage of the SNAP-23 peptide, and some reduced cleavage kinetics of the SNAP- 25 peptide.
  • FIG. 6C shows an expanded view of the activity modified BoNT/A light chains (LC) showing activity with the Chimera 3 and Chimera X10 chimeric peptide substrates.
  • FIG. 6D shows the results of additional screening studies, which identified additional mutations showing significant proteolytic activity with the Chimera 3 chimeric peptide substrate.
  • FIG. 6E shows the results of kinetic studies of cleavage of the Chimera 3 chimeric peptide substrate, which appear to be bi- modal.
  • compositions and methods that provide for screening of protease activity, particularly the activity of genetically modified proteases with modified specificity.
  • a series of recombinant peptide substrates and/or reporting constructs are provided that incorporate portions of both a parent substrate peptide (i.e. a peptide substrate of the native or wild-type protease) and portions of a target substrate peptide (i.e. a desired peptide substrate of the recombinant protease).
  • a parent substrate peptide i.e. a peptide substrate of the native or wild-type protease
  • a target substrate peptide i.e. a desired peptide substrate of the recombinant protease.
  • Members of a series of recombinant or chimeric peptide substrates and/or reporting constructs can provide different ratios and/or arrangements of parent substrate to target substrate content.
  • Recombinant proteases can be initially screened against a recombinant/chimeric peptide substrate and/or reporting construct having a cleavage site with a relatively high ratio of parent substrate content to target substrate content. Recombinant proteases identified in the initial round can be further screened using a different
  • the screening process can be repeated using recombinant/chimeric peptide substrates and/or reporting constructs with progressively lower parent substrate content until a recombinant protease is identified with the desired activity and/or specificity towards the target substrate.
  • Recombinant/chimeric peptide substrates can include identifiable tags, immobilizing/affinity tags, and other features that facilitate detection of protease activity.
  • inventive subject matter provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
  • Coupled to is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms “coupled to” and “coupled with” are used synonymously.
  • Inventors have noted that in preliminary stages of the development of genetically modified proteases with altered specificity, screening using only a peptide substrate sequence corresponding to the target protein can be ineffective in identifying genetically modified proteases with partial function. Accordingly, Inventors have developed an approach in which a series of chimeric peptide substrates and/or reporting constructs are provided. Such a series of chimeric peptide substrates and/or reporting constructs can include elements or portions of both a parent substrate (i.e. a peptide substrate of a native or wild type protease) and a target substrate (i.e. a peptide substrate representing the desired target of the genetically modified protease).
  • a parent substrate i.e. a peptide substrate of a native or wild type protease
  • a target substrate i.e. a peptide substrate representing the desired target of the genetically modified protease
  • a chimeric peptide substrate can include 1, 2, 3, 4, 5, or more recognition sites (i.e. peptide sequences that are recognized by and/or facilitate binding to a protease) of a parent substrate and/or target substrate, and/or 1 or more cleavage sites (i.e. peptide sequences where peptide cleavage occurs during proteolysis) of a parent substrate and/or target substrate.
  • recognition sites i.e. peptide sequences that are recognized by and/or facilitate binding to a protease
  • cleavage sites i.e. peptide sequences where peptide cleavage occurs during proteolysis
  • Such parent substrate and target substrate elements can be arranged in a“block” manner (e.g. all parent substrate sequences arranged sequentially and contiguously without intervening target substrate sequences) in a chimeric peptide substrate.
  • parent substrate and target substrate elements can be interspersed in a recombinant peptide substrate.
  • a series of chimeric peptide substrates and/or reporting constructs is prepared, each member of the series having different parent substrate and target substrate content and/or arrangement.
  • a chimeric peptide substrate and/or reporting construct utilized early in the screening process can contain more parent substrate content than target substrate content.
  • Genetically modified proteases that show activity with such a chimeric substrate and/or reporting construct can be further screened using a different chimeric peptide substrate and/or reporting construct with reduced parent substrate content, or using a series of chimeric peptide substrates and/or reporting constructs with incrementally reduced parent substrate contant.
  • findings from initial screening using chimeric peptide substrates and/or reporting constructs with high parent substrate content can be used to generate additional genetically modified proteases that are subsequently screened using chimeric peptide substrates and/or reporting constructs with less parent substrate content.
  • a chimeric peptide substrates can have a parent peptide substrate content that is 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10- fold, 7-fold, 5-fold, 3-fold, equivalent, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 7%, 5%, 3%, 1%, or less than 1% of the target peptide substrate content in a given chimeric peptide substrate and/or reporting construct.
  • Such ratios can be in terms of amino acids within the peptide, number of recognition sites, number of cleavage sites, and/or number of recognition and cleavage sites.
  • a chimeric peptide substrate and/or reporting construct that includes 2 recognition sites from a target peptide substrate and 3 recognition sites and a cleavage site from a parent peptide substrate can be characterized as having 40% target peptide content (i.e. 2 of 5 recognition sites) and 60% parent peptide substrate content (i.e. 3 of 5 recognition sites).
  • Activity of genetically modified proteases with chimeric peptide substrates and/or reporting constructs can be evaluated using any suitable method.
  • cleavage of a chimeric peptide substrate and/or reporting construct can be characterized by sizing techniques (e.g. SDS-PAGE, mass spectrometry, size exclusion chromatography, etc.) that identify the presence of one or more relatively lower molecular weight products of proteolysis.
  • a chimeric peptide substrate and/or reporting construct can include a detectable label (e.g.
  • a detectable label can be positioned at or near a terminus of the chimeric peptide substrate and/or reporting construct.
  • a chimeric peptide substrate and/or reporting construct can be coupled (reversibly or non-reversibly) to a solid phase such that the detectable label is released into solution on proteolysis.
  • the amount of detectable label in solution (or, alternatively, the relative lack of detectable label remaining on the solid phase) can be utilized to determine if proteolytic activity is present.
  • a chimeric peptide substrate and/or reporting construct can include an anchoring group (for example, biotin or a biotin analog) that facilitates coupling to the solid phase.
  • an anchoring group can be positioned at or near a terminus of the chimeric peptide substrate and/or reporting construct that is distal to the detectable label.
  • detectable labels can, for example, provide a direct visualization of activity (e.g. by disruption of FRET by separation of a FRET pair of fluorophores) or be used for immobilization/affinity capture (e.g. bead pull down, panning, etc.).
  • the chimeric peptide substrate and/or reporting construct can incorporate two or more labeling groups that interact, such that a lack of interaction following proteolysis results in a detectable change in an observable signal from the recombinant peptide substrate and/or reporting construct.
  • this can be provided using a pair of fluorophores that exchange energy (e.g. a FRET pair) or a fluorophore/quencher pair.
  • fluorophores and/or quenching groups can be provided by chemical modification of the chimeric peptide substrate, for example by reaction with reactive fluorescent dyes and/or quenchers.
  • such labeling groups can be provided as part of the chimeric peptide substrate and/or reporting construct during protein synthesis, for example by the inclusion of green fluorescent protein and/or green fluorescent protein mutant peptides.
  • the portions of parent substrate and target substrate would be interposed between the two or more labeling groups.
  • the genetically modified protease is derived from a Botulinum neurotoxin (i.e. a BoNT).
  • BoNTs utilize a number of spatially distinct substrate recognition sites for targeted binding in addition to specific cleavage site sequences. Suitable BoNTs include BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, and BoNT/G.
  • the parent substrate can be a conventional target of the respective BoNT.
  • the parent substrate can be SNAP-25 or a portion thereof that incorporates recognition and/or cleavage sites for BoNT/A or BoNT/E.
  • the parent substrate can be synaptobrevin or a portion thereof that incorporates recognition and/or cleavage sites for BoNT/B. It should be appreciated that in such embodiments a BoNT can be selected for genetic modification on the basis of the degree of the parent substrate’s homology to the desired target sequence.
  • BoNT/A can be utilized as a starting point for the genetically modified protease on the basis of the degree of homology between SNAP-23 or SNAP-29 with SNAP-25.
  • FIG. 1 An example of a series of chimeric reporting constructs that incorporate chimeric peptide substrates useful for screening BoNT/A light chain mutations for activity with a target SNAP-23 peptide substrate is shown in schematically in FIG. 1 (as well as a reporting peptide based on SNAP-25).
  • SNAP-25 includes 4 regions that act as recognition sites for the BoNT/A light chain protease (labeled 1 through 4), and a cleavage site (labeled 5) where peptide backbone scission occurs.
  • the chimeric protein shown at the top of FIG. 1 represents an embodiment of a peptide reporter carrying a fully SNAP-25 (i.e. parent) substrate peptide.
  • Two different mutant green fluorescent protein peptides are positioned at the termini of the substrate peptide and form a FRET pair. Cleavage of the intervening peptide, for example by BoNT/A, results in an observable loss of FRET.
  • a reporting construct can include one or more fluorescent moiety(ies) that are grafted onto or conjugated with the peptide following synthesis.
  • recombinant peptide substrate can include a fluorophore and a quenching moiety, such that cleavage of the reporting construct results in an increase in the observed fluorescence.
  • the chimeric peptide substrate and/or reporting construct can be captured on a solid phase.
  • the reporting construct can include a capture/affinity moiety that permits immobilization to a solid phase at one terminus and a detectable moiety on the remaining terminus.
  • Suitable capture/affinity moieties include biotin, iminobiotin, avidin or avidin derivatives, streptavidin or streptavidin derivatives, polyhistidine, haptens, and/or any suitable affinity moiety.
  • Suitable detectable moieties include fluorophores, luminescent groups, phosphorescent groups, enzymes, mass labels, microparticles, and radioactive isotopes.
  • Suitable solid phases include wells of a microwell plate, membranes, slides, filters, beads, and/or microparticles. Suitable microparticles can be encoded (for example, by the inclusion of one or more fluorescent dyes) to permit multiplex detection.
  • Such solid phases can incorporate complementary binding partners for the capture moiety.
  • cleavage can be performed when the corresponding reporting construct is in solution, or following capture on the solid phase.
  • the reporting construct can be coupled directly to the solid phase, either covalently or noncovalently.
  • the chimeric peptide substrate and/or reporting construct can lack a capture moiety.
  • the second reporting construct from the top of FIG. 1 represents a similar reporting construct in which the chimeric protein substrate includes only recognition and cleavage site portions from a target substrate peptide, in this instance SNAP-23.
  • SNAP-23 a series of chimeric reporting constructs can be provided with varying degrees of SNAP-25 and SNAP-23 content.
  • a reporting construct utilized in preliminary screening can have a majority of SNAP- 25 (i.e. parent) peptide substrate content. For example, only a single recognition site from SNAP-23 can be included.
  • Subsequent reporting constructs can include chimeric peptide substrate portions with increasing amounts of SNAP-23 content, for example including 2 recognition sites originating from SNAP-23, 2 recognition sites originating from SNAP-25, and a cleavage site from SNAP-25.
  • Still subsequent reporting constructs can include chimeric peptide substrate portions with a majority SNAP-23 content, for example 3 recognition sites originating from SNAP-23, a single recognition site from SNAP-25, and a SNAP-25 cleavage sites.
  • recognition sites are being shown as replaced in a serial fashion along the length of the chimeric peptide substrates, such sites can be replaced in a non- sequential or scattered fashion.
  • a parent peptide substrate cleavage site can be exchanged for a target peptide substrate cleavage site at any suitable point within such a series of chimeric peptide substrates.
  • FIG. 1 depicts a series of chimeric peptide substrates and/or reporting constructs in which increasing amounts of SNAP-23 content are provided at one terminus of a SNAP-25 peptide fragment
  • SNAP-23 sequences that surround select portions of native substrate (i.e. SNAP-25) peptide fragments can be utilized. Examples of such refined reporting constructs, as well as their relationship to SNAP23 and SNAP-25 amino acid sequences and the Botulinum neurotoxin serotype A cleavage site, are shown in FIG. 2.
  • such a series of reporting constructs can be used to screen a library of mutant proteases, for example a series of amino acid substitutions introduced into a BoNT/A light chain.
  • the sites of such mutations can be determined using enzyme/substrate docking studies.
  • a single library of mutant proteases is screened using a given series of reporting constructs containing chimeric peptide substrates.
  • results from an initial set of screening studies can provide guidance for the generation of additional members of a mutant protease library.
  • results of screening a library of mutant proteases containing single mutations can suggest additional mutant proteases that include combinations of such single mutations, which can be added to the protease library screened against subsequent reporting constructs containing chimeric peptide substrate portions with greater target peptide substrate content.
  • a typical set of experiments of such a screening study is shown in Table 1.
  • FIGs. 3A, 3B, and 3C Examples of reporting construct sequences and structures of the inventive concept for a series suitable for screening of BoNT/A light chain mutations with activity for SNAP-23 are shown in FIGs. 3A, 3B, and 3C.
  • FIGs. 3A to 3C include sequentially arranged blocks of peptide sequence derived from parent and target proteins, it should be appreciated that portions of peptide sequence from two or more substrate proteins can be interspersed within a cleavage region. It should also be appreciated that while FIGs. 3A to 3C depict hybrid peptide substrate portions incorporating portions of the sequence of a single substrate protein, such peptides can include portions of two or more substrate proteins.
  • a chimeric peptide substrate sequence can include one or more portions derived from a naturally occurring substrate protein and one or more portions that represent artificial sequences.
  • artificial sequences include consensus sequences and sequences derived from molecular modeling and/or docking simulations.
  • Reporting constructs as described above can be utilized in screening assays directed to identifying activity in a systematic series of mutations occurring at predetermined sites in a series of recombinant proteases (e.g. light chain or FC portions of Botulinum neurotoxins).
  • recombinant proteases e.g. light chain or FC portions of Botulinum neurotoxins
  • reporting constructs that include chimeric peptide substrates can be utilized in evolutionary approaches to generating proteases with modified substrate specificity.
  • An example of such an evolutionary approach is depicted in FIG. 4.
  • a library of BoNT/FC mutations can be generated by a suitable process, such as error-prone PCR, random mutagenesis, and or site-directed mutagenesis in combination with a suitable expression system.
  • Suitable expression systems include phage display and incorporation into an expression vector followed by transfection.
  • Proteolytic activity of members of such a library can be characterized using one or more reporting constructs that each incorporate a chimeric peptide substrate, followed by
  • binding activity can be determined by coupling a reporting construct incorporating a chimeric peptide substrate to a solid phase (or a series of such reporting constructs to a series of solid phases), and the resulting solid phase(s) used in a pull-down assay with sequence-modified BoNT/LC(s).
  • sequence-modified BoNT/LCs can be used in such a pull down assay as a mixture, and desirable mutations identified by sequencing following release from the solid phase.
  • a mutated protease e.g. a mutated BoNT LC/A
  • a mutated protease e.g. a mutated BoNT LC/A
  • the clone expressing the mutated protease can be used as the basis for additional rounds of mutagenesis and screening.
  • chimeric peptide substrates can be used in‘panning’ or similar assays to identify organisms displaying sequence-modified proteases (e.g. sequence-modified BoNT LC/As).
  • a chimeric peptide substrate (or a peptide that incorporates the chimeric peptide substrate) is coupled to an insoluble phase, such as a plate or a population of beads (e.g. magnetically responsive beads).
  • Cells displaying sequence-modified BoNT LC/As are brought into contact with the coated solid phase, then washed to remove unbound or loosely bound cells.
  • wash solutions of varying stringency can be used in order to select for cells with greater or lesser binding affinity.
  • Bound cells can then be recovered, for example from a plate surface or by selectively recovering cell-bearing beads using a cell sorter or similar device.
  • BoNT LC/A sequences having the desired binding affinity and/or substrate specificity can then be identified by sequencing the isolated clones.
  • clones so identified can be returned to the process and subjected to more stringent washing conditions in order to identify those with greater and lesser affinity and/or specificity. It should be appreciated that such a method is best suited for screening BoNT LC/A mutations with reduced or absent proteolytic activity.
  • chimeric peptide substrate sequences utilized in this fashion can be constructed to incorporate recognition and/or binding sites but exclude or modify the site at which proteolysis occurs.
  • results of exemplary screening studies of various BoNT LC/A mutations at the E148 site using a SNAP-25 based reporting construct, a SNAP-23 based reporting construct, and various chimeric reporting constructs are provided in FIG. 6A to 6E.
  • such studies have identified an EusM mutation in the Botulinum neurotoxin serotype A light chain as having enhanced proteolysis kinetics with the a reporting construct that incorporates the Chimera 3 chimeric peptide substrate, which includes primarily SNAP-23 recognition sites with a small portion of SNAP-25 that includes the cleavage site.
  • Additional active mutations of BoNT LC/A are apparent, including Ei4sC, Ei4sL, Ei4sV, and EusY.

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Abstract

L'invention concerne des compositions et des procédés permettant de cribler l'activité de protéases génétiquement modifiées pour identifier une spécificité de substrat modifié. Une série de substrats peptidiques recombinés sont générés qui incorporent à la fois des parties d'un peptide substrat parent (c'est-à-dire un substrat peptidique de la protéase native ou de type sauvage) et des parties d'un peptide substrat cible (c'est-à-dire un substrat peptidique souhaité de la protéase recombinée). Des protéases recombinées sont initialement criblées contre un substrat de peptide recombiné avec une quantité relativement élevé de contenu de substrat parent par rapport au contenu de substrat cible. Des protéases recombinées identifiées dans le criblage initial peuvent être ensuite criblées à l'aide de différents substrats peptidiques recombinés ayant une quantité inférieure de contenu de substrat parent par rapport au un contenu de substrat cible. Le processus de criblage peut être répété à l'aide de substrats peptidiques recombinés ayant un contenu de substrat parent progressivement inférieur jusqu'à ce qu'une protéase recombinée soit identifiée avec l'activité et/ou la spécificité souhaitées vers le substrat cible. Les substrats peptidiques recombinés peuvent comprendre des marqueurs identifiables, des marqueurs d'immobilisation/d'affinité, et d'autres caractéristiques facilitant la détection de l'activité protéase.
PCT/US2020/018089 2019-02-13 2020-02-13 Compositions et procédés de criblage de protéase WO2020168061A1 (fr)

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