Highly sensitive drug-screening method to find cruzain inhibitors from natural products by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy

This study describes a drug-screening methodology that screens cruzain inhibitors from natural products by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). Recombinant cruzain was expressed in Luria-Bertani Broth (LB) as an active mature protease using the existing E. coli bacterial expression system. The standard operating procedure for protein expression and purification was used. The limits of detection (LOD) were determined for commercially available cruzain inhibitors, leupeptin and E64, on the Agilent 1100 LC-MS system. The LOD reported here was compared to other LC-MS screening methods for natural products. Sample preparation, enzyme concentration, injection volume, flow rate, column format, ionization interface (both electrospray ionization and atmospheric pressure chemical ionization), detector settings, etc., were evaluated and optimized. The centrifuge filter, Microcon YM-10, which is used to remove the cruzain and inhibitor-cruzain complex, may cause a serious loss of active chemicals in the ultrafiltration process. Therefore, an internal standard, verbenone, was introduced into the system for calibration purposes. Various concentrations of artificial mixtures, such as simple inhibitors, multiple inhibitors, and multiple inhibitors with plant extracts were tested to verify the capability and efficiency of the screening method. To extend the LC-MS screening and fluorometric assay and possibly overcome some of their limitations, nuclear relaxation rate (T1) measurements were performed to identify specific and non-specific interactions between leupeptin and cruzain. The binding interaction between leupeptin and cruzain was observed. The activity of cruzain in the artificial samples containing simple, commercially available inhibitors was confirmed by fluorometric assays.