Cleavage of VEGF and VEGF receptor by wildtype and mutant MT-SP1

a technology of vegf receptor and vegf protein, which is applied in the field of vegf and vegf protein cleavage by wildtype and mutant mtsp1 and can solve the problems that no natural proteases are known to function, and achieves enhanced specificity toward target substrates, little to no activity, and reduced toxicity.

Inactive Publication Date: 2009-02-19
VERTEX PHARMA INC
View PDF70 Cites 30 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While it has been shown that some proteases are down regulated in cancer, to date no natural proteases are known to function in defending the body from tumorogenesis.

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
  • Cleavage of VEGF and VEGF receptor by wildtype and mutant MT-SP1
  • Cleavage of VEGF and VEGF receptor by wildtype and mutant MT-SP1
  • Cleavage of VEGF and VEGF receptor by wildtype and mutant MT-SP1

Examples

Experimental program
Comparison scheme
Effect test

example 1

Methods of Cloning and Characterizing Engineered MT-SP1 Protease with Altered Substrate Specificity Based on Well Understood Starting Scaffolds

[0191]The serine protease MT-SP1 has been chosen as scaffold protease for mutagenesis towards specific proteolysis of VEGF and VEGFR in part because it has been well characterized with biochemical and structural techniques [Harris, Recent Results Cancer Res. 1998; 152:341-52].

[0192]MT-SP1 is a membrane bound serine protease with multiple extracellular protein-protein interaction domains. The protease domain alone has been profiled using the totally diverse and P1-Lys PSSCL (FIG. 4A-4C) revealing an extended specificity of (basic)-(non-basic)-Ser-Arg or (non-basic)-(basic)-Ser-Arg / Lys. The X-ray crystallographic structure of MT-SP1 reveals components proposed to regulate activity and a nine amino acid insertion in the 60's loop that may determine P2 specificity

[0193]Variants of MT-SP1 have been created and characterized. Various protease mutei...

example 2

Expression and Purification of MT-SP1

[0194]A mutated MT-SP1 polypeptide (“mutein”) may contain a single mutation per polypeptide, or may contain two or more mutated residues in any combination, as illustrated in Table 11.

[0195]Wild-type and mutant MT-SP1 are cloned into the pQE bacterial expression vector (Qiagen) containing an N-terminal 6 histidine tag, prodomain, and protease domain and the resulting constructs transformed into BL21 E. coli cells. Cells are grown in 100 mL cultures to an OD of 0.6, and expression of the protease in inclusion bodies is induced by adding IPTG to a final concentration of 1 mM. After 4-6 hours, the bacteria are pelleted by centrifugation and the pellet resuspended in 50 mM Tris pH 8, 500 mM KCl, and 10% glycerol (buffer A). Cells are lysed by sonication and pelleted by centrifugation at 6000×g. Pellets are resuspended in 50 mM Tris pH 8, 6 M urea, 100 mM NaCl and 1% 2-mercaptoethanol (buffer B). Membrane and organelles are pelleted by centrifugation ...

example 3

Synthesis and Screening of Combinatorial Libraries for Characterization of MT-SP1 Wild-Type and Muteins

Fixed P1 Amino Acid Method

[0197]Individual P1-substituted Fmoc-amino acid ACC-resin (ca. 25 mg, 0.013 mmol) was added to wells of a Multi-Chem 96-well reaction apparatus. The resin-containing wells were solvated with DMF (0.5 mL). A 20% piperidine in DMF solution (0.5 mL) was added followed by agitation for 30 min. The wells of the reaction block were filtered and washed with DMF (3×0.5 mL). In order to introduce the randomized P2 position, an isokinetic mixture (Ostresh, J. M., et al., (1994) Biopolymers 34:1681-9) of Fmoc-amino acids (4.8 mmol, 10 equiv / well; Fmoc-amino acid, mol %: Fmoc-Ala-OH, 3.4; Fmoc-Arg(Pbf)-OH, 6.5; Fmoc-Asn(Trt)-OH, 5.3; Fmoc-Asp(O-t-Bu)-OH, 3.5; Fmoc-Glu(O-t-Bu)-OH, 3.6; Fmoc-Gln(Trt)-OH, 5.3; Fmoc-Gly-OH, 2.9; Fmoc-His(Trt)-OH, 3.5; Fmoc-Ile-OH, 17.4; Fmoc-Leu-OH, 4.9; Fmoc-Lys(Boc)-OH, 6.2; Fmoc-Nle-OH, 3.8; Fmoc-Phe-OH, 2.5; Fmoc-Pro-OH, 4.3; Fmoc-Ser...

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

PropertyMeasurementUnit
timeaaaaaaaaaa
pHaaaaaaaaaa
pHaaaaaaaaaa
Login to view more

Abstract

MT-SP1 mutein proteases with altered specificity for the target molecules they cleave can be used to treat human diseases, such as cancer. Cleaving VEGF or VEGFR at certain substrate sequences with wild-type and mutein MT-SP1 proteases can be used to treat pathologies associated with angiogenesis.

Description

RELATED APPLICATIONS[0001]This application is a divisional of U.S. application Ser. No. 11 / 104,110, filed Apr. 12, 2005, entitled “CLEAVAGE OF VEGF AND VEGF RECEPTOR BY WILDTYPE AND MUTANT MT-SP1” to Ruggles et al., which claims the benefit of priority to U.S. provisional application Ser. No. 60 / 561,720, filed Apr. 12, 2004, entitled “CLEAVAGE OF VEGF RECEPTOR BY WILDTYPE AND MUTANT MTSP1” to Ruggles et al. The subject matter of each of the above-noted applications is incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]The process of angiogenesis is central to the pathology of conditions including malignancy, diabetic retinopathy and macular degeneration. That cancer is angiogenesis-dependent has been recently supported by experimentation in which striking inhibition of tumor growth can be achieved not by direct treatment of the tumor, but rather by selective inhibition of the endothelial growth factor Vascular Endothelial Growth Factor (VEGF). VEGF is an endo...

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(United States)
IPC IPC(8): A61K51/00A61K38/48A61P31/00A61K39/395A61K38/00C12N9/64
CPCC12N9/50C12N9/6424A61P3/10A61P5/00A61P9/00A61P9/10A61P27/02A61P29/00A61P31/00A61P35/00A61P43/00C12N9/64
Inventor RUGGLES, SANDRA WAUGHNGUYEN, JACK
Owner VERTEX PHARMA INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap