Neprilysin is a transmembrane
zinc metallopeptidase that degrades a wide range of
peptide substrates. It has received attention as a potential
therapy for
Alzheimer's disease due to its ability to degrade the
peptide amyloid beta. However, its broad range of
peptide substrates has the potential to limit its
therapeutic use due to degradation of additional
peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of
neprilysin with improved activity and specificity towards
amyloid beta as a potential therapeutic for
Alzheimer's disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the
enzyme and variants screened for activity on
amyloid beta 1-40, 1-42 and a variety of other physiologically relevant
peptides. We identified several mutations that modulated and improved both
enzyme selectivity and intrinsic activity.
Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on
amyloid beta 1-40 and up to a 3,200-fold reduction in activity on other
peptides. Along with the altered
peptide substrate specificity, the mutant
enzyme produced a markedly altered series of
amyloid beta cleavage products compared to the wild-type
enzyme. Crystallisation of the mutant
enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type
enzyme. The mutant
enzyme offers the potential for the more efficient degradation of
amyloid beta in vivo as a therapeutic for the treatment of
Alzheimer's disease.