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. 2008 Dec 25;51(24):7737-43.
doi: 10.1021/jm800765e.

Interactions between human glutamate carboxypeptidase II and urea-based inhibitors: structural characterization

Cyril Barinka  1 , Youngjoo Byun, Crystal L Dusich, Sangeeta R Banerjee, Ying Chen, Mark Castanares, Alan P Kozikowski, Ronnie C Mease, Martin G Pomper, Jacek Lubkowski
Affiliations

Interactions between human glutamate carboxypeptidase II and urea-based inhibitors: structural characterization

Cyril Barinka et al. J Med Chem. .

Abstract

Urea-based, low molecular weight ligands of glutamate carboxypeptidase II (GCPII) have demonstrated efficacy in various models of neurological disorders and can serve as imaging agents for prostate cancer. To enhance further development of such compounds, we determined X-ray structures of four complexes between human GCPII and urea-based inhibitors at high resolution. All ligands demonstrate an invariant glutarate moiety within the S1' pocket of the enzyme. The ureido linkage between P1 and P1' inhibitor sites interacts with the active-site Zn(1)(2+) ion and the side chains of Tyr552 and His553. Interactions within the S1 pocket are defined primarily by a network of hydrogen bonds between the P1 carboxylate group of the inhibitors and the side chains of Arg534, Arg536, and Asn519. Importantly, we have identified a hydrophobic pocket accessory to the S1 site that can be exploited for structure-based design of novel GCPII inhibitors with increased lipophilicity.

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Figures

Figure 1
Figure 1
Chemical structures of NAAG and urea-based GCPII inhibitors.
Figure 2
Figure 2
Structural similarity of the glutamate binding in the S1′ pocket. The X-ray structures of rhGCPII/EPE (PDB code 3bi0), rhGCPII/glutamate complex (PDB code 2c6g), and the rhGCPII/2 complexes are superimposed using corresponding Cα-atoms. The active site ligands are in stick representation, residues shaping the S1′ pocket are shown as lines, Zn2+ ions as blue spheres, and conserved water molecules as red spheres. The H-bonds are indicated by dashed lines with distances shown in Ångstroms (from the rhGCPII/2 complex).
Figure 3
Figure 3
(A) Superposition of urea-based inhibitors in the active-site of rhGCPII. The rhGCPII–inhibitor complexes were superimposed on corresponding Cα-atoms. The inhibitors are shown in stick representation and protein residues are shown as lines. Note invariant positioning of the P1′ glutamate contrasting with inhibitor conformational variability in the S1 pocket. (B) Hydrogen-bonding network in the S1 site of GCPII. Hydrogen bonding interactions (indicated with dashed lines) and distances (in Ångstroms) between 1 bound in the active site and S1 residues of GCPII. The zinc ions, chloride anion, and water molecules located in the active site are shown as blue, yellow and red spheres, respectively. The protein and inhibitor atoms are colored red (oxygen), blue (nitrogen), yellow (sulfur), violet (iodine), cyan (fluorine), gray (carbons).
Figure 4
Figure 4
The hydrophobic pocket accessory to the S1 site. The dissected substrate-binding cavity of GCPII is shown in semitransparent surface representation (gray). The side chains of amino acids delineating the “accessory hydrophobic pocket” are shown in stick representation and colored cyan. The active-site Zn2+ and S1-bound Cl− are colored blue and yellow, respectively, and water molecules are represented by red spheres. Compound 4 bound to the active site is in stick representation.
Figure 5
Figure 5
The flexibility of S1 arginines 463 and 536 defines size of the “accessory pocket”. Dissected view into the active-site of GCPII. The protein moiety is shown in combination of cartoon and line representation. The residues shaping walls of the accessory pocket are shown as spheres and inhibitor (substrate) residues are in stick representation. The active site Zn2+ and S1 bound Cl− are colored blue and yellow, respectively. The R463u and R463d denotes the side chain of Arg463 in the “up” and “down” position, respectively, while R536b and R536s denotes the side chain of Arg536 in the “binding” and “stacking” configuration, respectively. Notice the closure of the accessory pocket in the unliganded GCPII structure (A). Shown are complexes of rhGCPII with 2 (C), 1 (D), 3 (E), and 4 (F). The complex between the E424A active-site mutant of GCPII and NAAG, the GCPII natural substrate, is included for comparison (PDB code 3bxm, (B)).
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