Binding of alkaloids into the S1 specificity pocket of alpha-chymotrypsin: Evidence from induced circular dichroism spectra

Non-covalent binding of planar aromatic molecules into the S1 specificity pocket of the serine protease a-chymotrypsin (alpha CHT) can be detected by measuring induced circular dichroism (CD) spectroscopic signals. Utilizing this phenomenon, alpha CHT association of proflavine (PRF), the well known serine protease inhibitor has been investigated together with plant-derived compounds including isoquinoline, pyridocarbazole and indoloquinoline alkaloids, of which alpha CHT binding has never been reported. Non-degenerate exciton coupling between pi-pi* transitions of the ligand molecules and two tryptophan residues (Trp172 and Trp215) near to the binding site is proposed to be responsible for the induced CD activity. The association constants calculated from CD titration data indicated strong alpha CHT association of sanguninarine, ellipticine, desmethyl-isocryptolepine and isoneocryptolepine (K-a approximate to 10(5) M-1) while berberine, coptisine and chelerythrine bind to the enzyme with lower, PRF-like affinity (K-a approximate to 10(4) M-1). PRF-trypsin and ellipticine-trypsin binding interactions have also been demonstrated. The binding of the alkaloids into the S1 pocket of alpha CHT has been confirmed by CD competition experiments. Molecular docking calculations showed the inclusion of PRF as well as the alkaloid molecules in the S1 cavity where they are stabilized by hydrophobic and H-bonding interactions. These novel nonpeptidic scaffolds can be used for developing selective inhibitors of serine proteases having chymotrypsin-like folds. Furthemore, the results provide a novel, CD spectroscopic based approach for probing the ligand binding of alpha CHT and related proteases.