Crystal structures of complexes of GCPII with P1’-diversified urea-based inhibitors

Jiri Pavlicek,¹ Jakub Ptacek,¹ Jiri Cerny,¹ Youngjoo Byun,^2,3 Lubica Skultetyova,¹ Martin G. Pomper,² Jacek Lubkowski,⁴ and Cyril Barinka¹

¹Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague 4, Czech Republic

²Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical Institutions, 1550 Orleans Street, Baltimore, MD 21231

³College of Pharmacy, Korea University, 2511 Sejong-ro, Sejong, 339-700, South Korea

⁴Center for Cancer Research, Frederick National Laboratory for Cancer Research, Macromolecular Crystallography Laboratory, Frederick, MD 21702, USA

Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging prostate cancer and its metastases. In parallel efforts, agents with increased lipophilicity are designed and evaluated for targeting GCPII residing in the neuronal compartment. Here we report the structural and computational characterization of six complexes between GCPII and P1’-diversified urea-based inhibitors that have the C-terminal glutamate replaced by a more hydrophobic moiety. Our results provide a detailed description of interactions governing the recognition of non-glutamate residues in the S1’ pocket of the enzyme. We observed the flexibility of the S1’ pocket, most notably that of Phe209, Leu428, and Lys699 side chains, as well as the structural rearrangement of the amino acid segment Leu259 – Gly263, that allows GCPII to accommodate non-glutamate moieties at the S1’ site. The X-ray structures are complemented by the quantum mechanics calculations that provide a quantitative insight into the GCPII/inhibitor interactions. Presented data can be used for the rational design of novel glutamate-free GCPII inhibitors with improved physicochemical properties.