Design of Highly Potent Urea-Based, Exosite-Binding Inhibitors Selective for Glutamate Carboxypeptidase II

Tykvart, Jan; Schimer, Jiří; Jančařík, Andrej; Bařinková, Jitka; Navrátil, Václav; Starková, Jana; Šrámková, Karolína; Konvalinka, Jan; Majer, Pavel; Šácha, Pavel

doi:10.1021/acs.jmedchem.5b00278

Cited by 28 publications

(26 citation statements)

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“…The Asn to Ser substitution enables two conformations of the Zn2-coordinating amino acid Asp443 and is thus likely responsible for the lower occupancy of the Zn2 ion within the GCPIII active site [21], while the Trp to Lys substitution likely disrupts the ABS [7,20]. Disruption of the ABS in GCPIII can be exploited for development of inhibitors that are highly specific towards GCPII [34]. We corroborated these results by showing that endogenous substrates such as FolGlu n , which utilize the ABS in GCPII [7], have substantially lower affinity (higher K M value) towards GCPIII compared to GCPII.…”

Section: Discussionmentioning

confidence: 99%

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

et al. 2016

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Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave b-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DatabaseThe X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09. Abbreviations 3D, three-dimensional; AAS, atomic absorption spectroscopy; ABS, arene-binding site; ACN, acetonitrile; BCG, b-citryl-L-glutamic acid (bcitrylglutamate, beta-citrylglutamate, beta-citryl-L-glutamic acid, beta-citryl-L-glutamate); BSA, bovine serum albumin; C12E8, octaethylene glycol monododecyl ether; DBU, 1,8-Diazabicycloundec-7-ene; DIEA, N,N-Diisopropylethylamine; EtOAc, ethylacetate; FolGlu n , folyl-n-c-Lglutamic acid; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; GCPII, glutamate carboxypeptidase II; GCPIII, glutamate carboxypeptidase III; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; MeOH, methanol; MD/ MM, molecular dynamical/molecular mechanical calculations; NAAG, N-acetyl-L-aspartyl-L-glutamic acid; NMR, nuclear magnetic resonance; OPA, orthophtalaldehyde; ORF, open reading frame; Pd(C), palladium on activated charcoal; PSMA, prostate-specific membrane antigen; QM/MM, quantum mechanical and molecular mechanical calculations; QM/MM/MD, quantum mechanical, molecular mechanical and molecular dynamical calculations; qPCR, quantitative polymerase chain reaction; rhGCPII, recombinant human glutamate carboxypeptidase II; SDS/PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TLC, thin-layer chromatography.

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Section: Discussionmentioning

confidence: 99%

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

et al. 2016

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“…This result raises the question whether binding to a substance other than PSMA might be a possible contributing factor. Natural candidate proteins would include PSMA homologs, such as glutamate carboxypeptidase III (21,22) or N-acetylated a-linked acidic dipeptidaselike protein (23). Further insight could be gained by performing tracer uptake assays on tumor cell lines, therefore excluding the effect of tracer binding to neovasculature.…”

Section: Discussionmentioning

confidence: 99%

⁶⁸Ga-PSMA-HBED-CC PET for Differential Diagnosis of Suggestive Lung Lesions in Patients with Prostate Cancer

et al. 2015

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In prostate cancer (PC) patients, the differentiation between lung metastases and lesions of different origin, for example, primary lung cancer, is a common clinical question. Herein, we investigated the use of Glu-NH-CO-NH-Lys(Ahx)-HBED-CC ( 68 Ga-PSMA-HBED-CC) for this purpose. Methods: PC patients (n 5 1,889) undergoing 68 Ga-PSMA PET/CT or PET/MR scans were evaluated retrospectively for suggestive lung lesions. For up to 5 lesions per patient, location, CT diameter, CT morphology, and SUV max were determined. The standard for classification was either histopathologic evaluation or, in the case of PC metastases, responsivity to antihormone therapy. A comparison of the different classes was executed by Student t test. Prostate-specific antigen and prostate-specific membrane antigen (PSMA) immunohistochemistry were performed if histologic samples were available; 68 Ga-PSMA autoradiography was performed on an exemplary case of PET-positive lung cancer. Results: Eighty-nine lesions in 45 patients were identified, of which 76 were classified as PC (39 proven, 37 highly probable), 7 as primary lung cancer, and 2 as activated tuberculosis; 4 lesions remained unclear. The mean SUV max was 4.4 ± 3.9 for PC metastases and 5.6 ± 1.6 for primary lung cancer (P 5 0.408). Additionally, substantial differences in SUV max intraindividually were detected. The 2 tuberculous lesions showed an SUV max of 7.8 and 2.5. Using immunohistochemistry, we could demonstrate PSMA expression in the neovasculature of several PSMA PET-positive lung cancers as well as in tuberculous lesions from our histologic database. Conclusion: Quantitative (SUV) analysis of 68 Ga-PSMA PET was not able to discriminate reliably between pulmonary metastases and primary lung cancer in PC patients. The reason for the unexpectedly high tracer uptake in non-PC lesions is not completely clear. PSMA expression in neovasculature provides a possible explanation for this finding; however, other contributing factors, such as tracer binding to proteins other than PSMA, cannot be excluded at present.

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“…Additionally, it facilitates the engagement of these moieties with structurally defined pockets in the entrance funnel (e.g., the S1 accessory hydrophobic pocket or the arene-binding site), thus contributing to the increased affinity of such bivalent ligands for PSMA ( Fig. 6) and allowing for the structure-assisted design of the next generation of ligands (48,(51)(52)(53)(54)(55).…”

Section: Lesson 2: Need For Structure-aided Design Of Glu-ureido-basementioning

confidence: 99%

Glu-Ureido–Based Inhibitors of Prostate-Specific Membrane Antigen: Lessons Learned During the Development of a Novel Class of Low-Molecular-Weight Theranostic Radiotracers

et al. 2017

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Design of Highly Potent Urea-Based, Exosite-Binding Inhibitors Selective for Glutamate Carboxypeptidase II

Cited by 28 publications

References 36 publications

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

⁶⁸Ga-PSMA-HBED-CC PET for Differential Diagnosis of Suggestive Lung Lesions in Patients with Prostate Cancer

Glu-Ureido–Based Inhibitors of Prostate-Specific Membrane Antigen: Lessons Learned During the Development of a Novel Class of Low-Molecular-Weight Theranostic Radiotracers

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Design of Highly Potent Urea-Based, Exosite-Binding Inhibitors Selective for Glutamate Carboxypeptidase II

Cited by 28 publications

References 36 publications

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

68Ga-PSMA-HBED-CC PET for Differential Diagnosis of Suggestive Lung Lesions in Patients with Prostate Cancer

Glu-Ureido–Based Inhibitors of Prostate-Specific Membrane Antigen: Lessons Learned During the Development of a Novel Class of Low-Molecular-Weight Theranostic Radiotracers

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⁶⁸Ga-PSMA-HBED-CC PET for Differential Diagnosis of Suggestive Lung Lesions in Patients with Prostate Cancer