A Novel Screen of PAD4 Inhibitors

Reference #: 00689

Invention Description:

The subject invention is an Activity-Based Protein Profiling (ABPP) assay in which a potential inhibitor compound competes with rhodamine-conjugated fluoroamidine to bind to PAD4. Fluorescence is measured to determine an estimate of the amount of fluorescent protein arginine deiminase 4 that is present in the assay.

Potenttial Applications:

Protein Arginine Deiminase 4 (PAD4) is a leading target for the development of a Rheumatoid Arthritis (RA) pharmaceutical.

Advantages and Benefits:

• Overcomes the limitations of commonly used PAD4 assays by eliminating the need for strong acids, high temperatures, and toxic compounds

• Allows for the direct visualization of PAD4 inhibition

Background:

While additional PAD activity assays have recently been described in the literature (e.g., an ELISA based assay that monitors PAD4 activity through the use of an anti-citrulline antibody28 and a coupled assay that couples the production of ammonia to the glutamate dehydrogenase catalyzed reductive amination of α-ketoglutarate29), these assays provide only an indirect measure of PAD4 activity. In contrast, this assay allows for the direct visualization of PAD4 inhibition.

Furthermore, this invention represents the first use of a competitive ABPP assay to overcome the limitations of previous assays in a system with purified proteins, thereby providing an additional application for ABPP reagents and suggesting a general application for ABPP assays in drug discovery.