Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

VARIANTS OF AMYLOID [beta]-PROTEIN PRECURSOR INHIBITOR DOMAIN

A technology of phenylalanine and leucine, applied in protease inhibitors, in vivo radioactive preparations, peptide/protein components, etc., can solve the problem of the absence of trypsin in humans

Inactive Publication Date: 2018-10-23
THE NAT INST FOR BIOTECH IN THE NEGEV LTD
View PDF8 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] There are particular challenges in developing inhibitors that will target mid-trypsin, especially since this enzyme is resistant to inhibition by many polypeptide serine protease inhibitors and further cleaves and inactivates many of these inhibitors as physiological substrates
An additional challenge exists from the need for selective inhibitors, since trypsin in mesotrypsin exhibits an affinity for the major digesting trypsins (cationic and anionic trypsins), as well as other serine proteases, including kallikrein and coagulation factors. High sequence homology and structural similarity
Thus, it is not surprising that there are currently no potent inhibitors with high stability, affinity and specificity for trypsin in humans

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • VARIANTS OF AMYLOID [beta]-PROTEIN PRECURSOR INHIBITOR DOMAIN
  • VARIANTS OF AMYLOID [beta]-PROTEIN PRECURSOR INHIBITOR DOMAIN
  • VARIANTS OF AMYLOID [beta]-PROTEIN PRECURSOR INHIBITOR DOMAIN

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0210] APPI displayed by yeast WT Rapidly cleaved by human trypsin

[0211] The yeast surface display (YSD) system for directed evolution is based on the expression of a library of mutant proteins on the surface of yeast, followed by selection of variants with improved affinity. However, this system has not previously been used to identify proteolytic cleavage or improve proteolytic resistance of displayed inhibitors. In order to test APPI WT Compatibility with YSD system, APPI WT The coding region of was cloned into the YSD plasmid for presentation on the surface of S. cerevisiae as a fusion with the Aga2p lectin protein. APPI was then validated using FACS by detecting bound fluorescently labeled bovine trypsin, which is a tight binding target of established APPI WT correct folding. Such as Figure 1A As seen in , APPI displayed on the surface of yeast is highly expressed and shows significant binding to bovine trypsin, which exhibits proper folding of APPI ( Figure 1A ...

Embodiment 2

[0214] Strategies for Proteolytic Stability Maturation of APPI Libraries

[0215] Motivated by the discovery that active mid-trypsin proteolyzes surface-displayed APPI and that mid-trypsin-S195A can detect residual, uncleaved APPI on the cell surface, it was hypothesized that these reagents could be used in a stepwise fashion to enrich for proteolytically resistant APPI diversity library of variants. As a starting point, a random library was generated in which mutations were introduced throughout the APPI gene at a frequency of 0-3 mutations / clone, which yielded approximately 9B10 6 A library of independent variants. Diversity is introduced throughout the molecule because, while protease specificity is primarily directed by the sequence of the canonical binding loop, proteolytic stability was previously found to be a property strongly influenced by residues within the scaffold.

[0216] The unique screening strategy presented, designated "triple staining", consists of three ...

Embodiment 3

[0219] High affinity and high stability variants identified at S5

[0220] Show APPI WT 'Triple staining' analysis of cells from and from library maturation cycles (S1 to S5) showed that the more advanced the sorting, the higher the stability and affinity of trypsin ( Figure 2B 1 ); notable was S5, which showed high resistance to the proteolytic activity of medium trypsin at all enzyme concentrations used. After generating a library comprising resistant clones, the inventors proceeded to determine whether it would be possible to detect binding interactions between active trypsins (as done using bovine trypsin) for each stability maturation screening step. Indeed, 'double staining' analysis of cells from categories S1 to S5 with active mid-trypsin (without inactive mid-trypsin) showed high binding in late sorting rounds (i.e., S4, S5, Figure 2B 2 ). These results indicate a relatively high population of proteolysis-resistant APPI variants (ie, stability-matured variants)...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Variants of amyloid [beta]-protein precursor inhibitor domain (A PPI), effective in inhibiting mesotrypsin and / or kallikrein-6, and composition comprising same, are provided. Further, methods of use of the peptides or composition, including, but not limited to treatment of cancer are provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 265,719, filed December 10, 2015, and U.S. Provisional Patent Application No. 62 / 313,824, filed March 28, 2016, the contents of which are incorporated by reference in their entirety This article. technical field [0003] The present invention relates to peptides derived from the amyloid ∮-protein precursor inhibitor domain (APPI) effective in inhibiting specific serine proteases such as mesotrypsin and / or kallikrein-6, and uses thereof Methods, including but not limited to treating malignant diseases, and the like. Background technique [0004] Human amyloid∮-protein precursor inhibitor (APPI), also known as protease connexin-2, is a secreted form of amyloid∮-protein precursor (APP). APPI contains a Kunitz serine protease inhibitor domain known as KPI (Kunitz Protease Inhibitor). [0005] Serine proteases are enzymes that c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81A61K38/55A61P35/00G01N33/48
CPCC07K14/81C12N9/50A61K38/00A61K51/088C07K14/8114A61P35/04A61K38/55A61K51/08
Inventor N·帕波I·科恩A·萨纳诺斯
Owner THE NAT INST FOR BIOTECH IN THE NEGEV LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com