Method for identification of protease activity inhibitors and assaying the presence of protease activity

Abstract

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.

Description

CROSSREFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61 / 267,386, entitled “BOTULINUM NEUROTOXIN INHIBITOR IDENTIFICATION METHOD AND SYSTEM” and filed Dec. 7, 2009, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]This invention generally relates to the field of protease inhibitor identification assays.BACKGROUND[0003]Proteases play an important role in biological processes. Proteases, however, can also cause significant harm to biological systems particularly those delivered by virus, toxins and pathogenic micro-organisms. Methods for developing protease inhibitors and assaying for protease activity particularly in cells is a critical area of biotechnology. For example, the Botulinum neurotoxins (BoNTs) are the most potent toxins known (S. S. Arnon, R. Schechter, et al. Jama 285:1059-70. (2001); and B. M. Paddle. J Appl Toxicol 23:139-70. (2003). Botulism can b...

AI Technical Summary

Problems solved by technology

Proteases, however, can also cause significant harm to biological systems particularly those delivered by virus, toxins and pathogenic micro-organisms.

A negative consequence of their usefulness is the increased availability of the neurotoxins for misuse Likewise, increased usage increases the likelihood of the occurrence of unintended adverse effects during treatment.

As such, long-term mechanical ventilation would be impractical if even a limited number of individuals were simultaneously affected.

Once diaphragm muscles are affected, breathing is impaired and ultimately suffocation results.

Therefore, the active sites differ sufficiently among the serotypes, such that broad-spectrum potential inhibitors are unlikely.

However, identification and inoculation of all members of large at risk populations prior to exposure is problematic.

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