title: Probing secondary glutaminyl cyclase (QC) inhibitor interactions applying an in silico-modeling/site-directed mutagenesis approach: implications for drug development.
abstract: Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate-modified amyloid peptides deposited in neurodegenerative disorders such as Alzheimer's disease. Inhibitors of QC are currently in development as potential therapeutics. The crystal structures of the potent inhibitor PBD150 bound to human and murine QC (hQC, mQC) have been described recently. The binding modes of a dimethoxyphenyl moiety of the inhibitor are significantly different between the structures, which contrasts with a similar K(i)  value. We show the conformation of PBD150 prone to disturbance by protein-protein interactions within the crystals. Semi-empirical calculations of the enzyme-inhibitor interaction within the crystal suggest significant differences in the dissociation constants between the binding modes. To probe for interactions in solution, a site-directed mutagenesis on hQC was performed. The replacement of F325 and I303 by alanine or asparagine resulted in a 800-fold lower activity of the inhibitor, whereas the exchange of S323 by alanine or valine led to a 20-fold higher activity of PBD150. The results provide an example of deciphering the interaction mode between a target enzyme and lead substance in solution, if co-crystallization does not mirror such interactions properly. Thus, the study might provide implications for rapid screening of binding modes also for other drug targets.
authors.affiliation: Department of Enzymology, Probiodrug AG, Weinbergweg 22, 06120 Halle (Saale), Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Koch
authors.forename: Mirko
authors.initials: M
authors.lastname: Buchholz
authors.forename: Michael
authors.initials: M
authors.lastname: Wermann
authors.forename: Ulrich
authors.initials: U
authors.lastname: Heiser
authors.forename: Stephan
authors.initials: S
authors.lastname: Schilling
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Demuth
mesh_terms: D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D051379:Mice; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase