title: Engineering neprilysin activity and specificity to create a novel therapeutic for Alzheimer's disease.
abstract: 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.
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Cambridge, United Kingdom.
authors.forename: Carl I
authors.initials: CI
authors.lastname: Webster
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Cambridge, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Burrell
authors.affiliation: Discovery Sciences, AstraZeneca R & D, Mölndal, Sweden.
authors.forename: Lise-Lotte
authors.initials: LL
authors.lastname: Olsson
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Cambridge, United Kingdom.
authors.forename: Susan B
authors.initials: SB
authors.lastname: Fowler
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Cambridge, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Digby
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Cambridge, United Kingdom.
authors.forename: Alan
authors.initials: A
authors.lastname: Sandercock
authors.affiliation: Discovery Sciences, AstraZeneca R & D, Mölndal, Sweden.
authors.forename: Arjan
authors.initials: A
authors.lastname: Snijder
authors.affiliation: Global Drug Discovery, Global Biologics, Bayer HealthCare AG, Cologne, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Tebbe
authors.affiliation: Global Drug Discovery, Global Biologics, Bayer HealthCare AG, Cologne, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Haupts
authors.affiliation: Global Drug Discovery, Global Biologics, Bayer HealthCare AG, Cologne, Germany.
authors.forename: Joanna
authors.initials: J
authors.lastname: Grudzinska
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Cambridge, United Kingdom.
authors.forename: Lutz
authors.initials: L
authors.lastname: Jermutus
authors.affiliation: CNS&P iMed, AstraZeneca R & D, Södertälje, Sweden.
authors.forename: Christin
authors.initials: C
authors.lastname: Andersson
mesh_terms: D000544:Alzheimer Disease / Q000188:drug therapy; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015260:Neprilysin / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D015260:Neprilysin
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