Synaptic Research, LLC

Other

The United States of America, As Represented by the Secretary, Department of Health and Human Services

Ipsen Pharma S.a.s.

Yien Che Tsai

George A Oyler

Paul S Fishman

Randall L Kincaid

Yung-Nien Chang

Allan M Weissman

Bo Chen

Jennifer Mariano

A system for the identification of proteases and protease inhibitors is provided. The system has at least two components. The first component is a reporter construct with at least one binding site, a transcriptional promoter, an inducible promoter region, and at least one reporter gene, all functionally connected for expression of the reporter gene(s) in functional coordination with a transcriptional activation agent. The second component is a transcriptional activation agent comprising a nucleic acid binding domain, at least one protease substrate domain, and at least one transcriptional activation domain for an inducible promoter. The system allows detection and evaluation of agents affecting protease activity directed to the protease substrate domain. The system also allows for the detection of the presence of proteases in environmental samples.

System for the assessment of protease activity

A cell based assay for detection for protease activity is disclosed. In the assay a cell is engineered to express a protease substrate with at least one label, preferably on its C-terminus. Cleavage of the substrate by the protease that recognizes it results in a C-terminal fragment and a N-terminal fragment, where the fragment having the label is subject to ubiquitin proteasome degradation. The assay measures the disappearance of the label due to degradation of the fragment to which it is attached. A cell free assay is also described for detection of protease activity. In the cell free assay, the protease substrate is expressed in a solution that includes the elements of the ubiquitin proteasome pathway for degradation of the fragment. The assay measures the disappearance of the label attached to the fragment that results from cleavage by the protease.

N-end rule protease activity indication methods and uses thereof

The present invention relates to a designer or recombinant ubiquitin ligase molecule that includes a toxin binding domain that is specific for a toxin active fragment, wherein the toxin active fragment is an enzymatically active fragment of one or more toxins or toxin serotypes; and an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin active fragment. In an embodiment, the composition further includes a delivery system that allow the designer ubiquitin ligase to enter the cell. The present invention further includes methods for treating an individual intoxicated with a toxin by administering the designer ubiquitin ligase of the present invention.

Methods of degrading or inhibiting botulinum neurotoxin using designer ubiquitin ligases

A system and method for identifying aneurotoxin inhibitor employing aneurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. Theneurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express aneurotoxin substrate complex. Also provided are novel stable cell lines that express thetoxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

Methods for identifying inhibitors of botulinum neurotoxins

The present invention features isolated nucleic acid molecules, designated G2BD nucleic acid molecules, which encode the binding site from the gp78 ubiquitin ligase that binds to the Ube2G2 ubiquitin conjugating enzyme. The invention further provides isolated G2BD proteins and fusion proteins. Still further provided are diagnostic and therapeutic methods, as well as screening assays utilizing compositions of the invention.

Ube2G2 binding domain in the ubiquitin ligase gp78 and methods of use thereof

Designer ubiquitin ligases having a non-cleavable SNAP25 domain and E3-ligase domain

A system and method for identifying a botulinum neurotoxin inhibitor employing a botulinum neurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. The botulinum neurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express a botulinum neurotoxin substrate complex. The methods of the present invention are adapted for cell based screening to monitor the catalytic activity of a BoNT in living cells and to identify molecules that inhibit the catalytic activity of a BoNT in living cells. Also provided are novel stable cell lines that express the botulinum toxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

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Frederick, MD, United States of America

Yien Che Tsai