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

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.

SUBMITTER: Barinka C

PROVIDER: S-EPMC5516903 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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

Barinka Cyril C Byun Youngjoo Y Dusich Crystal L CL Banerjee Sangeeta R SR Chen Ying Y Castanares Mark M Kozikowski Alan P AP Mease Ronnie C RC Pomper Martin G MG Lubkowski Jacek J

Journal of medicinal chemistry 20081201 24

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' ...[more]

PMID: 19053759

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Project description:Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics. Our data show that higher binding affinities of phosphoramidates, compared to matching phosphonates, are linked to the presence of additional hydrogen bonds between Glu424 and Gly518 of the enzyme and the amide group of the phosphoramidate. While the positioning of the P1' glutamate-derived module within the S1' pocket of GCPII is invariant, interaction interfaces between effector functions and residues lining the entrance funnel are highly varied, with the positively charged arginine patch defined by Arg463, Arg534 and Arg536 being the only 'hot-spot' common to several studied complexes. This variability stems in part from the fact that the effector/GCPII interfaces generally encompass isolated areas of nonpolar residues within the entrance funnel and resulting van der Waals contacts lack the directionality typical for hydrogen bonding interactions. The presented data unravel a complexity of binding modes of inhibitors within non-prime site(s) of GCPII and can be exploited for the design of novel GCPII-specific compounds.Atomic coordinates of the present structures together with the experimental structure factor amplitudes were deposited at the RCSB Protein Data Bank under accession codes 4P44 (complex with JRB-4-81), 4P45 (complex with JRB-4-73), 4P4B (complex with CTT54), 4P4D (complex with MP1C), 4P4E (complex with MP1D), 4P4F (complex with NC-2-40), 4P4I (complex with T33) and 4P4J (complex with T33D).

Project description:BackgroundGlutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets.MethodsIdentified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species.ResultsSingle genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice.ConclusionsOur study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

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

Publications

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

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