Inhibitory activity of amino acid derivatives against native Cryptococcus neoformans urease and ligand-enzyme binding...

Studies on ligand-enzyme binding modes on functional microorganism ureases are currently unexplored mainly due to the difficulties in obtaining purified native or recombinant microbial ureases. The investigation of interactions with small ligands, kinetic parameters and most of inhibitor screening is usually performed using the commercially available plant urease model. Herein, the unique urease paramagnetic Ni(II) bimetallic center and saturation transfer from protein to ligand protons were explored by NMR to access the ligand-urease binding profile in Cryptococcus neoformans cell lysates. Thiohydantoins derived from L-valine (1a), D-valine (1b) and L-methionine (3) presented high inhibitory percentages on the ureolytic activity of the fungal lysates, ranging from 84.0 % to 86.3 %. Evaluation of the relaxation rate of the water protons by NMR during titration with inhibitor 1a, revealed the binding of the ligand to the Ni(II) catalytic site of the fungal urease in the lysates. The enhanced relaxation rate of the water protons resonance, induced by their contact with the paramagnetic urease site, decreased linearly from 0.7 s^-1 to 0.4 s^-1 in response to the 1a additions, culminating, at 3.0 mM of 1a, the same value of the enzyme-free control solution (0.4 s^-1). The Saturation Transfer Difference (STD) analysis of ligand 1a with the fungal urease was reliably achieved by comparison of NMR experiments on lysates with high ureolytic activities and fungal lysates with vanished activity. The STD data showed an intimate contact of the side chain of the inhibitor to the C. neoformans urease.