Peptidase cleavable substrates and methods of identification and use thereof
AssigneeAlaunus Biosciences, Inc.
InventorsGiselle M. Knudsen, Mark D. Cisneros
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Abstract
The present disclosure relates to library compositions, methods of making and screening libraries of peptidase-cleavable substrate sequences. In particular, the libraries are useful in identifying substrate sequences for a variety of disease- or tissue-specific peptidases. The present disclosure also relates to use of identified peptidase substrates to design therapeutic agents and diagnostic tools.
Claims21
A method of constructing an unbiased library of peptides or proteins, comprising:
- i. designing a peptide library by a combination of biological assays and computational modeling wherein each peptide comprises at least a set of two, three or more amino acid motifs;
- ii. generating a plurality of peptides comprising the at least a set of two, three or more amino acid motifs which are variable based on amino acid sequences and positioning of each motif in each peptide;
- iii. assessing cooperative interactions between at least two, three or more amino acid motifs within a given sequence space for a defined number of amino acid residues and length of a peptide substrate sequence;
- iv. extracting of amino acid motifs by a computational algorithm by measuring cooperativity between two or more unique amino acids found at specific positions within substrate sequences of a peptide library;
- thereby constructing an unbiased library of peptides or proteins.
A method of building peptidase cleavable substrate library is provided, the method comprises the steps of:
- (i) Providing an unbiased library of peptides or proteins, wherein the peptide or protein comprises a substrate sequence, and the design of the library is the result of mathematical modeling to assess all possible cooperative interactions between at least two, three or more amino acid residues within a given sequence space for a defined number of residues and length of substrate sequence;
- (ii) Incubating the library in the presence of the peptidase, allowing the peptidase to cleave peptides or proteins within the library to form a population of cleaved and non-cleaved peptides or proteins;
- (iii) Screening the population of cleaved and non-cleaved peptides or proteins for obtaining an amino acid motif(s) comprising a minimal set of two or more amino acids at varying positions within a peptidase enzyme substrate sequence that interact with the peptidase or peptidases (Gearr™ motifs);
- (iv) Extracting the amino acid motifs using a computational algorithm;
- and
- (v) Building a refined peptidase cleavable substrate sequence library from amino acid motifs, wherein the substrate sequences are capable of being cleaved by at least one peptidase.
19-21. (canceled)
Dependencies are inferred from the claim text.