Investigations using NMR spectroscopy to understand the binding of small molecules towards cystein proteases (papain, cruzain) to support bioassay results

This study describes the development of a drug screening methodology that monitors the intermolecular interaction of cysteine protease, Papain, and its inhibitor, Leupeptin by Nuclear Magnetic Resonance (NMR) spectroscopy. The limit of detection (LOD) for the detection of molecular interaction of Leupeptin with Papain was evaluated on a Varian 500 MHz INOVA NMR spectrometer equipped with a penta probe and VNMRJ 2.1B software. The binding of Leupeptin to Papain, as well as 2-Octanone and 2-Nonanone to Bovine Serum Albumin (BSA) was evaluated and significant spectral parameters determined. Sample preparation, BPPSTE and One-Shot DOSY methods, Non-selective Spin-Lattice Relaxation (T 1) method, selective Spin-Lattice Relaxation (T1SE ) method, and Spin-Spin Relaxation (T2) method were optimized and evaluated. Furthermore the preparation of cruzain was optimized to increase the yield and allow it to incorporated in future studies. Recombinant cruzain was expressed using an existing E. coli expression system and purified as an active cysteine protease.