WO2020168061A1

WO2020168061A1 - Compositions and methods for protease screening

Info

Publication number: WO2020168061A1
Application number: PCT/US2020/018089
Authority: WO
Inventors: Ward C. Tucker; Francis Mark DUNNING
Original assignee: Biomadison, Inc.
Priority date: 2019-02-13
Filing date: 2020-02-13
Publication date: 2020-08-20

Abstract

Compositions and methods are described that provide for screening the activity of genetically modified proteases to identify modified substrate specificity. A series of recombinant peptide substrates are generated that incorporate both portions of 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). Recombinant proteases are initially screened against a recombinant peptide substrate with a relatively high ratio of parent substrate content to target substrate content. Recombinant proteases identified in the initial screening can be further screened using a different recombinant peptide substrates with a lower ratio of parent substrate content to target substrate content. The screening process can be repeated using recombinant peptide substrates with progressively lower parent substrate content until a recombinant protease is identified with the desired activity and/or specificity towards the target substrate. Recombinant peptide substrates can include identifiable tags, immobilizing/affinity tags, and other features that facilitate detection of protease activity.

Description

COMPOSITIONS AND METHODS FOR PROTEASE SCREENING

[0001] This application claims the benefit of United States Provisional Patent Application No. 62/805,086 filed on February 13, 2019. These and all other referenced extrinsic materials are incorporated herein by reference in their entirety. Where a definition or use of a term in a reference that is incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein is deemed to be controlling.

Field of the Invention

[0002] The field of the invention is identification of protease activity, particularly from genetically modified proteases.

Background

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Enzymes, in particular proteases, have found use as therapeutic agents. For example, Botulinum neurotoxins (BoNTs) have been utilized for therapeutic purposes that exploit the remarkably specific catalytic activity of the protein’s light chain with SNAP-25 and other proteins utilized in neurotransmitter release. 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.

[0005] Genetic engineering offers the opportunity to modify the specificity and/or activity of proteases through the introduction of one or more mutations. The current state of the art, however, does not provide for accurate prediction of the effects of individual or multiple mutations of protease activity or specificity. While docking studies can direct an investigator towards sites where sequence modification may be fruitful, extensive screening of mutation libraries remains necessary to identify potentially useful mutants. While such screening studies (for example, as found in PCT Application Publication No. WO 2005/087947, to Gregg) can be performed using the desired substrate protein it should be appreciated that success using such an approach requires that one or more mutant(s) generated in an initial mutation library happens to have significant activity with the target protein. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Such an approach relies heavily on random chance, and as such even extensive screening can fail to provide a useful result.

[0006] Attempts have been made to address this issue. For example, United States Patent No. 6,506,557, to Chadwick and Russell, describes an evolutionary approach where a chimeric peptide that includes an envelope protein necessary for viral replication and an inhibitory domain with an interposing substrate sequence is encoded on a plasmid. Expression of a protease within a transfected cell that cleaves the substrate sequence permits viral replication, providing selection. Such a method, however, requires the use of complex and expensive cell culture techniques, and places inherent limitations on the size of the intervening substrate sequence.

[0007] Thus, there is still a need for efficient and effective compositions and methods for identifying enzymes with modified activity.

Summary of The Invention

[0008] The inventive subject matter provides apparatus, systems and methods in which a—

[0009] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

Brief Description of The Drawings

[0010] FIG. 1: 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.

[0011] FIG. 2: 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.

[0012] FIGs 3A to 3C: 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. 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.

[0013] FIG. 4: FIG. 4 schematically depicts an exemplary evolutionary approach to modifying binding and/or substrate specificity of Botulinum neurotoxin serotype A light chain (BoNT/LC).

[0014] FIG. 5: FIG. 5 schematically depicts an exemplary screening assay for binding to chimeric peptide substrates of the inventive concept.

[0015] FIGs. 6A to 6E: FIG. 6A shows results of screening of BoNT LC/A mutations

MutOl (E148) through Mutl l (E148). As shown, no mutations resulted in cleavage of the SNAP-23 peptide, and some abolished cleavage of the SNAP-25 peptide. One clone showed novel kinetics with the Chimera 3 chimeric peptide substrate, and some increase in the rate of Chimera X10 chimeric peptide substrate cleavage was noted. 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. None of these clones showed cleavage of the chimeric peptide substrates. 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.

Detailed Description

[0016] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0017] The inventive subject matter provides 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). 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

recombinant/chimeric peptide substrate(s) and/or reporting constmct(s) with a lower ratio and/or different arrangement of parent substrate content and target substrate content relative to the recombinant/chimeric peptide substrate and/or reporting construct utilized for initial screening. 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.

Any suitable detection method that can identify reactivity with a recombinant/chimeric peptide substrate and/or reporting construct can be utilized (e.g. fluorescence, luminescence, molecular weight tag, affinity tag, hapten tag, etc.). Recombinant/chimeric peptide substrates can include identifiable tags, immobilizing/affinity tags, and other features that facilitate detection of protease activity.

[0018] One should appreciate that the disclosed techniques provide many advantageous technical effects including provision of a detectable level of activity in the screening of recombinant proteases during early stages of development, facilitating the use of evolutionary approaches to identifying recombinant proteases with new activities and/or specificities.

[0019] The following discussion 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.

[0020] As used herein, and unless the context dictates otherwise, the term "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.

[0021] As used in the description herein and throughout the claims that follow, the meaning of “a,”“an,” and“the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of“in” includes“in” and“on” unless the context clearly dictates otherwise.

[0022] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0023] 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).

[0024] For example, 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. 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. In other embodiments parent substrate and target substrate elements can be interspersed in a recombinant peptide substrate. Such parent substrate elements and/or target substrate elements can range from 1 to 10, 20, 30 50, 75, 100, 150, 200, or more amino acids in length.

[0025] In a preferred embodiment of the inventive concept 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. For example, 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. In some embodiments, findings from initial screening using chimeric peptide substrates and/or reporting constructs with high parent substrate content (e.g, >50%)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.

[0026] 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. For example, 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).

[0027] Activity of genetically modified proteases with chimeric peptide substrates and/or reporting constructs can be evaluated using any suitable method. For example, 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. In other embodiments, a chimeric peptide substrate and/or reporting construct can include a detectable label (e.g. a fluorophore, lumiphore, enzymatic label, affinity tag, etc.), where proteolytic activity provides a detectable change in the observable properties and/or distribution of the detectable label. Such a detectable label can be positioned at or near a terminus of the chimeric peptide substrate and/or reporting construct. For example, 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. In such embodiments 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. Such 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. Such 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.).

[0028] In some embodiments of the inventive concept 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. In a preferred embodiment of the inventive concept this can be provided using a pair of fluorophores that exchange energy (e.g. a FRET pair) or a fluorophore/quencher pair. Such 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. Alternatively, 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. In such chimeric peptide substrates and/or reporting constructs the portions of parent substrate and target substrate would be interposed between the two or more labeling groups.

[0029] In some embodiments of the inventive concept 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. In such embodiments the parent substrate can be a conventional target of the respective BoNT. For example, for a genetically modified protease derived from BoNT/A or BoNT/E the parent substrate can be SNAP-25 or a portion thereof that incorporates recognition and/or cleavage sites for BoNT/A or BoNT/E. Similarly, for a genetically modified protease derived from BoNT/B 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. For example, if SNAP-23 and/or SNAP-29 are the desired target substrates, 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.

[0030] 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). As noted above, 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 (Cyan Fluorescent Protein or CFP and Yellow Fluorescent Protein or YFP) 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.

[0031] Although depicted in FIG. 1 as including a pair of green fluorescent protein-derived fluorophores selected and positioned to act as a FRET pair, it should be appreciated that other configurations are also suitable. In some embodiment a reporting construct can include one or more fluorescent moiety(ies) that are grafted onto or conjugated with the peptide following synthesis. In other embodiments 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.

[0032] In some embodiments of the inventive concept the chimeric peptide substrate and/or reporting construct can be captured on a solid phase. In such embodiments 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.

[0033] Such solid phases can incorporate complementary binding partners for the capture moiety. In methods employing such chimeric peptide substrates cleavage can be performed when the corresponding reporting construct is in solution, or following capture on the solid phase. In some embodiments the reporting construct can be coupled directly to the solid phase, either covalently or noncovalently. In such embodiments the chimeric peptide substrate and/or reporting construct can lack a capture moiety.

[0034] Similarly, 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. To identify mutations in a BoNT/A light chain that have activity with SNAP-23 a series of chimeric reporting constructs can be provided with varying degrees of SNAP-25 and SNAP-23 content.

As shown, 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. It should be appreciated that, while 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. Similarly, 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.

[0035] It should be appreciated that as modified enzyme sequences are identified with modified specificity, chimeric peptide substrates and/or reporting constructs can be further refined. For example, while 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.

[0036] As noted above, 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. In some embodiments a single library of mutant proteases is screened using a given series of reporting constructs containing chimeric peptide substrates. In other embodiments, results from an initial set of screening studies can provide guidance for the generation of additional members of a mutant protease library. For example, 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.

Table 1

[0037] 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. Although the examples in 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. In some embodiments 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. Examples of artificial sequences include consensus sequences and sequences derived from molecular modeling and/or docking simulations.

[0038] 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).

Alternatively, 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. As shown, 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. [0039] 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

determination of the degree or extent of cleavage of each of the reporting constructs. For example, if the chimeric peptide substrate sequences are interposed between members of a FRET pair of fluorophores changes in FRET emission can be used. Alternatively, 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). In some embodiments 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.

[0040] In some embodiments, when a mutated protease (e.g. a mutated BoNT LC/A) is identified that shows an improvement in the desired enzyme activity, binding activity, and/or substrate specificity the clone expressing the mutated protease can be used as the basis for additional rounds of mutagenesis and screening.

[0041] An alternative binding assay utilizing chimeric peptide substrates as described above is shown in FIG. 5. As shown, 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). In such embodiments 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. In some embodiments 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. In some embodiments 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. Alternatively, 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.

[0042] 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. For example, 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. Without wishing to be bound by theory, the Inventors believe that activity with the Chimera 3 chimeric substrate peptide can be conferred by replacement of Ei4s with an amino acid having a non-polar aliphatic side chain, with the exception of EusA and Ei4sG.

[0043] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and“comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMS What is claimed is:

1. A method of characterizing protease activity, comprising:

providing a plurality of chimeric peptide substrates wherein each of the plurality of

chimeric peptide substrates comprises a first content comprising at least a portion of a parent peptide substrate and a second content comprising at least a portion of a target peptide substrate;

contacting a candidate protease with a first chimeric peptide substrate of the plurality of chimeric peptide substrates wherein the first content of the parent peptide substrate exceeds the second content of the target peptide substrate; characterizing a first cleavage activity of the candidate protease with the first chimeric peptide substrate;

contacting the candidate protease with a second chimeric peptide substrate of the plurality of chimeric peptide substrates wherein the second content of the target peptide substrate exceeds the first content of the parent peptide substrate; characterizing a second cleavage activity of the candidate protease with the second

chimeric peptide substrates; and

comparing the first cleavage activity with the second cleavage activity.

2. The method of claim 1, wherein the candidate protease is a genetically modified BoNT/A light chain (LC).

3. The method of claim 1 or 2, wherein the first content of the parent peptide substrate comprises a cleavage site or a recognition site.

4. The method of one of claims 1 to 3, wherein the second content of the target peptide substrate comprises a cleavage site or a recognition site.

5. The method of one of claims 1 to 4, wherein the parent peptide substrate comprises at least a portion of SNAP-25.

6. The method of one of claims 1 to 5, wherein the target peptide substrate is selected from the group consisting of SNAP-23 and SNAP-29.

7. The method of one of claims 1 to 6, wherein the first chimeric peptide substrate and the second chimeric peptide substrate are provided as a portion of a first chimeric reporting construct and a portion of a second chimeric reporting construct, respectively.

8. The method of claim 7 wherein the first chimeric reporting construct further comprises a first FRET pair and the second chimeric reporting construct comprises a second FRET pair.

9. The method of claim 8, wherein at least one of the first or second FRET pair comprises a donor fluorophore and a quencher.

10. The method of claim 8 or 9, wherein cleavage of the first chimeric peptide substrate is indicated by disruption of FRET in the first chimeric reporting construct.

11. The method of claim 7, wherein at least one of the first chimeric reporting construct and the second chimeric reporting construct comprises an affinity tag.

12. The method of claim 11, where the method comprises a step of capturing the affinity tag on an insoluble phase.

13. The method of claim 12, wherein the insoluble phase is a test fixture surface or a suspended bead surface.

14. A method of screening a protease library, comprising:

obtaining a protease library comprising a first protease and a second protease;

contacting the first protease with a first chimeric peptide substrate of the plurality of chimeric peptide substrates wherein the first content of the parent peptide substrate exceeds the second content of the target peptide substrate; characterizing a first cleavage activity of the first protease with the first chimeric peptide substrate; contacting the first protease with a second chimeric peptide substrate of the plurality of chimeric peptide substrates wherein the second content of the target peptide substrate exceeds the first content of the parent peptide substrate; characterizing a second cleavage activity of the first protease with the second chimeric peptide substrate;

contacting the second protease with the first chimeric peptide substrate;

characterizing a third cleavage activity of the second protease with the first chimeric peptide substrate;

contacting the second protease with the second chimeric substrate peptide;

characterizing a fourth cleavage activity of the second protease with the second chimeric peptide substrate; and

comparing the first cleavage activity, second cleavage activity, third cleavage activity, and fourth cleavage activity.

15. The method of claim 14, wherein the first protease and the second protease comprise different genetically modified BoNT/A light chains.

16. The method of claim 14 or 15, wherein the first content of the parent peptide substrate comprises a cleavage site or a recognition site.

17. The method of one of claims 14 to 16, wherein the second content of the target peptide substrate comprises a cleavage site or a recognition site.

18. The method of one of claims 14 to 17, wherein the parent peptide substrate comprises at least a portion of SNAP-25.

19. The method of one of claims 14 to 18, wherein the target peptide substrate is selected from the group consisting of SNAP-23 and SNAP-29.

20. The method of one of claims 14 to 19, wherein the first chimeric peptide substrate and the second chimeric peptide substrate are provided as a portion of a first chimeric reporting construct and a portion of a second chimeric reporting construct, respectively.

21. The method of claim 20, wherein the first chimeric reporting construct further comprises a first FRET pair and the second chimeric reporting construct comprises a second FRET pair.

22. The method of claim 21, wherein at least one of the first or second FRET pair comprises a donor fluorophore and a quenche.

23. The method of claim 21 or 22, wherein cleavage of the first chimeric peptide substrate is indicated by disruption of FRET in the first chimeric reporting construct.

24. The method of claim 20, wherein at least one of the first chimeric reporting construct and the second chimeric reporting construct comprises an affinity tag.

25. The method of claim 24, where the method comprises a step of capturing the affinity tag on an insoluble phase.

26. The method of claim 25, wherein the insoluble phase is a test fixture surface or a suspended bead surface.

27. A chimeric reporting construct for characterizing protease activity comprising a chimeric peptide substrate, wherein the chimeric peptide substrate comprises a first content of a parent peptide substrate and a second content of a target peptide substrate, wherein the first content is selected from the group consisting of a recognition site of the parent peptide substrate and a cleavage site of the parent peptide substrate and the second content is selected from the group consisting of a recognition site of the target peptide substrate and a cleavage site of the target peptide substrate.

28. The chimeric reporting construct of claim 27, further comprising an observable tag.

29. The chimeric reporting construct of claim 27 or 28, further comprising an immobilization tag.

30. The chimeric reporting construct of one of claims 27 to 29, comprising a FRET pair.

31. The chimeric reporting construct of claim 30, wherein at least one member of the FRET pair comprises a green fluorescent protein peptide or a mutant green fluorescent protein peptide.

32. The chimeric reporting construct of claim 20, wherein the FRET pair comprises a donor fluorophore and a quencher.

33. The chimeric reporting construct of one of claims 27 to 32, wherein the parent peptide substrate comprises at least a portion of SNAP-25.

34. The chimeric reporting construct of one of claims 27 to 33, wherein the target peptide substrate comprises at least a portion of SNAP-23 or at least a portion of SNAP-29.

35. A library of chimeric peptide substrates for characterizing protease activity, comprising: a first chimeric peptide substrate comprising a first content of a parent peptide substrate and a second content of a target peptide substrate, wherein the first content is selected from the group consisting of a recognition site of the parent peptide substrate and a cleavage site of the parent peptide substrate and the second content is selected from the group consisting of a recognition site of the target peptide substrate and a cleavage site of the target peptide substrate, wherein the first content exceeds the second content; and

a second chimeric peptide substrate comprising a third content of the parent peptide substrate and a fourth content of the target peptide substrate, wherein the fourth content exceeds the third content.

36. The library of claim 35, wherein the first chimeric peptide substrate and the second chimeric peptide substrate are provided as part of a first reporting construct and part of a second reporting construct, respectively, wherein each of the first reporting construct and the second reporting construct further comprise an observable tag.

37. The library of claim 36, wherein the first chimeric reporting construct and the second chimeric reporting construct each comprise an immobilization tag.

38. The library of one of claims 35 to 37, wherein the first chimeric reporting construct and the second chimeric reporting construct each comprise a FRET pair.

39. The library of claim 38, wherein at least one member of the FRET pair comprises a green fluorescent protein peptide or a mutant green fluorescent protein peptide.

40. The library of claim 38, wherein the FRET pair comprises a donor fluorophore and a quencher.

41. The library of one of claims 35 to 40, wherein the parent peptide substrate comprises at least a portion of SNAP-25.

42. The library of one of claims 35 to 41, wherein the target peptide substrate comprises at least a portion of SNAP-23 or at least a portion of SNAP-29.