Tau protease compositions and methods of use
Inventors
Moe, James G. • Davidowitz, Eliot J. • Lopez, Patricia
Assignees
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Publication Number
US-10597649-B2
Publication Date
2020-03-24
Expiration Date
Abstract
Disclosed are mammalian tau proteases, as well as proteolytically-active fragments, variants, and mutants thereof. Also disclosed are polynucleotides and recombinant expression vectors that encode these polypeptides, as well as methods for producing such proteins in selected recombinant host cells, and for using the compositions in a variety of diagnostic and analytical assays.
Core Innovation
The invention relates to mammalian tau proteases and tau protease-active fragments, variants, mutants, and truncated tau protease peptides or polypeptides that retain substantial serine protease activity. Tau is described as having a role in AD pathology, and the document describes tau autoproteolytic cleavage, proteolytic regions, and tau protease fragments and cut sites, including primary cut sites such as 130KS131, 340KS341, 353KI354, and 395KS396 and additional cut sites defined by KS/KT/KI motifs.
The disclosed tau proteases include protease-active truncated tau regions and variants having substitutions that enhance or diminish serine protease activity compared with naturally occurring tau peptides or naturally occurring tau polypeptides. The document describes identifying tau autoproteolytic cut sites and delineating proteolytic regions using truncated constructs that retain serine protease activity, including exemplified constructs such as TP-210 and TP-99.
The disclosure further describes tau protease cleavage of peptide and protein substrates relevant to AD pathology, including α-endorphin and APP 667-676 with implications for Aβ(1-42) generation, and additional substrates such as tau monomer/dimer/oligomers, tubulin, and specific peptide substrates. It also describes immunodetection and diagnostic strategies that target tau protease, as well as assay formats including tau protease activity assays, inhibitor profiling, and FRET-based protease detection assays using FRET peptide substrates and probe or primer sets.
Claims Coverage
The provided independent claims cover three inventive features involving isolated truncated tau peptides or polypeptides and truncated tau protease variants with specified sequence requirements and serine protease activity thresholds relative to full-length Tau 4R2N trimer protease.
Isolated truncated tau peptides with drug-target configuration and altered serine protease activity
An isolated truncated tau peptide or polypeptide of 50 to 300 amino acids that comprises an amino acid sequence at least 98% identical to an at least 50-amino-acid contiguous sequence from SEQ ID NO:6; the amino acid sequence is SEQ ID NO:6; configured to be a drug target; comprises enhanced or diminished serine protease activity when compared to that of a naturally occurring tau peptide or a naturally occurring tau polypeptide; comprises at least one substitution at positions corresponding to specified residues of SEQ ID NO:6; and is expressed in a bacterial host cell.
Truncated tau protease variants with substitutions that enhance serine protease activity
A truncated tau protease variant peptide or polypeptide comprising an amino acid sequence of a starting mammalian tau protease that has been substituted in at least one amino acid at positions corresponding to specified residues of SEQ ID NO:6; the substitution confers enhanced serine protease activity compared to that of the original, un-substituted tau protease peptide or polypeptide; the amino acid sequence is SEQ ID NO:6; and the truncated tau protease variant peptide or polypeptide is expressed in a bacterial host cell.
Isolated truncated tau peptides with retained serine protease activity and bacterial expression
An isolated truncated tau peptide or polypeptide of 50 to 300 amino acids comprising an at least 40 contiguous amino acid sequence from SEQ ID NO:6; mutated at one or more amino acids at positions corresponding to specified residues of SEQ ID NO:6; the truncated tau peptide or polypeptide has serine protease activity at least 85% of that of the full-length Tau 4R2N trimer protease; and is expressed in a bacterial host cell.
Across the independent claims, the invention is directed to isolated truncated tau peptides/polypeptides and truncated tau protease variants that retain serine protease activity, with contiguous sequence constraints relative to SEQ ID NO:6, defined mutation positions, quantified retained or enhanced serine protease activity relative to the full-length Tau 4R2N trimer protease, and expression in a bacterial host cell.
Stated Advantages
Enhanced or diminished serine protease activity relative to naturally occurring tau peptides or naturally occurring tau polypeptides.
Retained serine protease activity at specified percentages relative to full-length Tau 4R2N trimer protease.
Configuration as a drug target.
Assay and analytical utility for detecting tau protease activity using diagnostic or analytical assay formats including FRET-based protease detection.
Documented Applications
Immunodetection, diagnosis, and monitoring in AD are described for tau protease detection strategies and biomarkers.
Diagnostic or analytical assessment of tau protease activity using diagnostic/analytical assay formats, including FRET-based protease detection assays with FRET peptide substrates and probe or primer sets.
Drug-target configuration of truncated tau peptides or polypeptides having enhanced or diminished serine protease activity.
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