The role of the non-pharmacophore pocket of glutamate carboxypeptidase II in the design of small-molecule inhibitors

Zora Nováková,¹ Clifford E. Berkman,² and Cyril Bařinka¹

¹Laboratory of Structural Biology, Institute of Biotechnology, AS CR, Prague

²Department of Chemistry, Washington State University, USA

Inhibitors targeting human glutamate carboxypeptidase II (GCPII) are used in various diagnostic and therapeutic applications and typically consist of a C-terminal docking moiety and a functionalized distal moiety linked via a zinc-binding group. While details of interactions between GCPII the C-terminal part of an inhibitor are reasonably well established, there is a limited amount of structural information related to interactions between GCPII and distal functionalities. Here we present a comprehensive structural study aimed at dissecting the impact of physicochemical characteristics of distal functionalities on GCPII binding/affinity. To this end we determined crystal structures of eight complexes between GCPII and phosphoramidate-based inhibitors featuring an invariant P1’ glutamate and a variable P1 distal functionality. While the positioning of the phosphoramidate-glutamate functionality in the GCPII binding site is identical for all compounds tested, there are profound differences in GCPII interactions with distal groups and those are reflected in varied affinities and inhibition modes. Our data unravel a complexity of different binding modes of inhibitors within the S1 site of GCPII and can be exploited for the design of novel GCPII-specific compounds.