Computer assisted Docking and Mutagenesis studies of Astacin Proteases and their Natural Inhibitors as a Basis for Drug Design

Astacin proteases catalyze key reactions in animal embryogenesis and tissue differentiation. Consequently, their malfunction may cause severe disorders rendering this group of enzymes therapeutic targets for cancer and neurodegenerative disorders like Alzheimer’s disease. As protein cleaving enzymes, astacins need to be tightly controlled by activators and inhibitors. Recently, we could resolve the structural basis for astacin activation. In the inactive pro-astacins, an intrinsic pro-peptide blocks accessfor substrates to the active site. The pro-peptide is not cleaved, because it binds in inverse orientation compared to a substrate. The interactions between astacin proteases and their substrates have been also elucidated with synthetic inhibitors, which are resistant to cleavage by astacins. In addition, we discovered naturally occurring proteins inhibiting astacin proteases in vivo. However, the mode of interaction of astacins with these inhibitors is completely unknown. With no structures of astacin-(protein)inhibitor complexes available, we will decipher the protease-inhibitor interactions by molecular in silico docking experiments using the available high-resolution X-ray crystal structures of astacins and pro-astacins and their inhibitory counterparts. In addition, we will verify these potential interaction sites in the wet lab by site directed mutagenesis and functional analysis by enzyme-inhibitor kinetics.