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Zymogen activators

A technology of zymogen activating peptides and zymogens, which can be used in growth factors/inducing factors, animal/human proteins, digestive systems, etc., and can solve problems such as binding affinity limitation, weak therapeutic potential activator peptides, etc.

Inactive Publication Date: 2014-08-20
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this establishes the ability to allosterically modulate pro-HGF signaling activity by targeting the activation pocket of HGFβ, the therapeutic potential of this approach is limited by the very weak binding affinity (KD ~ 2 mM) of the activator peptide (Landgraf , K.E. et al. (2010) J Biol Chem285:40362-72)

Method used

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Examples

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Effect test

Embodiment 1

[0278] Example 1: Phage display of a specific peptide library of zymogen-like scHGF with an activation pocket yields highly conserved sequence motifs

[0279] To identify peptides that specifically bind the activation pocket of the zymogen-like form of HGFβ (scHGFβ) with high affinity, based on the structural and sequence homology found in the N-terminal residues of the trypsin / chymotrypsin-like serine protease domain, Engineered phage peptide libraries. Interestingly, there is high structural homology between the activation pocket of the trypsin / chymotrypsin-like serine protease domain and that of HGFβ (Kirchhofer, D. et al. (2004) J Biol Chem 279:39915-24; Kirchhofer , D. et al. (2007) Proc Natl Acad Sci USA 104:5306-11), clearly highlighting the functional conservation of the N-terminal insertion mechanism. In addition to structural homology, the pfam (Finn, R.D. et al. (2010) Nucleic Acids Res38:D211-22) consensus sequence profile of the N-terminal residues frequently fou...

Embodiment 2

[0281] Example 2: A synthetic peptide based on a phage-derived consensus motif binds scHGFβ with high affinity and activates the binding of scHGFβ to Met

[0282] Based on the finding that the consensus ZAP contains features consistent with binding and insertion into the activation pocket, several related peptides were synthesized and used in a previously established scHGFβ activation assay to assess their affinity and ability to activate Met binding (Landgraf, K.E. et al. (2010) J Biol Chem 285:40362-72). Since scHGFβ interacts only weakly with the Met Sema-PSI domain compared to the active HGFβ form, biolayer interferometry was used in the activation assay to measure the direct enhancement of the peptide by scHGFβ on streptavidin coated with biotinylated Met. Avidin sensor surface binding affinity capacity (Landgraf, K.E. et al. (2010) J Biol Chem 285:40362-72). Thus, titration of the activator peptide should produce a significant increase in the surface response signal due...

Embodiment 3

[0293] Example 3: Synthetic peptides activate pro-HGF and stimulate cell survival

[0294] To test whether these new more potent peptide activators could also activate the full-length form of pro-HGF and stimulate Met signaling, the peptides were screened for activity in a cell survival assay. Uncleaved forms of pro-HGF (scHGF), which are inactive as Met ligands, have been shown to not stimulate cell survival under serum-starved medium conditions; however, activator peptides in combination with scHGF can signal via Met and greatly enhance cell survival, Similar to that of proteolytically activated HGF ligands (Landgraf, K.E. et al. (2010) J Biol Chem 285:40362-72). Peptide titration in the presence of scHGF was performed using Bx-PC3 cells and a robust activation of signaling via Met observed for scHGF was seen, reaching levels equivalent to HGF-induced signaling.

[0295] The IVGG.14 and IVdG.14 peptides exhibited dose-dependent activation of scHGF, EC 50 for ~20μM peptide ...

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Abstract

Provided herein are zymogen activating molecules such as zymogen activating peptides, and methods of identifying and using these zymogen activating molecules such as zymogen activating peptides.

Description

[0001] related application [0002] This application claims Provisional U.S. Application No. 61 / 547,628 filed October 14, 2011, U.S. Application No. 61 / 648,470 filed May 17, 2012, and June 18, 2012 under 35 U.S.C. § 119 The benefit of US Application No. 61 / 661,180, the contents of which are hereby incorporated by reference in their entirety. field of invention [0003] Provided herein are zymogen activating molecules, such as zymogen activating peptides, and methods of identifying and using these zymogen activating molecules, such as zymogen activating peptides. [0004] sequence listing [0005] This application contains a Sequence Listing submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on October 10, 2012, is named P4768R1WO_PCTSequenceListing.txt and is 66,227 bytes in size. Background of the invention [0006] Hepatocyte growth factor (HGF), also known as scatter factor, is a member of the plasminogen-related grow...

Claims

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Application Information

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IPC IPC(8): A61K38/18C07K7/06C07K14/475
CPCC07K14/4753C07K7/06A61P1/16A61P3/10A61P7/12A61P19/04
Inventor K.E.兰德格拉芙R.A.拉扎鲁斯
Owner F HOFFMANN LA ROCHE & CO AG
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