Boehringer Ingelheim International Gmbh

Boehringer Ingelheim (canada) Ltd.

Boehringer Ingelheim Pharma Gmbh & Co Kg

Roger Maurice

Diane Thibeault

Daniel Lamarre

Louise Pilote

Armin Pause

Diane Thibault

The invention provides a novel assay for NS2/3 cleavage detection, particularly, a novel assay for the detection of the NS3 cleavage product in the presence of uncleaved NS2/3 based on discrimination of the activity of the resulting NS3 protease product from the NS3 protease activity of the NS2/3 uncleaved protein. Certain reaction conditions allow this discrimination in situ without having to resort to physical separation of both proteases by decreasing the NS3 protease activity of the NS2/3 protein and increasing the NS3 protease activity of the resulting NS3 protease product, thereby producing a 'signal window' upon which it is feasible to distinguish and measure NS3 protease activity produced from the cleavage of NS2/3.

Hepatitis C virus NS2/3 activity assay

A method for producing a refolded, inactive form of recombinantly produced NS2/3 protease which comprises the steps of: a) purifying the protease from inclusion bodies in the presence of a chaotropic agent; and b) refolding the purified protease by contacting it with a reducing agent and lauryldiethylamine oxide (LDAO) in the presence of reduced concentration of chaotropic agent or polar additive. The invention further comprises a method for activating this refolded inactive NS2/3 protease by adding an activation detergent. This method produces large amounts of the active NS2/3 protease to allow small molecules and ligands to be screened as potential inhibitors of NS2/3 protease, which may be useful as therapeutic agents against HCV.

Purified active HCV NS2/3 protease

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Montreal, Canada

Roger Maurice