Identification of 2-PMPA as a novel inhibitor of cytosolic carboxypeptidases.

Cytosolic carboxypeptidases (CCPs) comprise a unique subfamily of M14 carboxypeptidases and are erasers of the reversible protein posttranslational modification- polyglutamylation. Potent inhibitors for CCPs may serve as leading compounds targeting imbalanced polyglutamylation. However, no efficient CCP inhibitor has yet been reported. Here, we showed that 2-phosphonomethylpentanedioic acid (2-PMPA), a potent inhibitor of the distant M28 family member glutamate carboxypeptidase II (GCPII), rather than the typical M14 inhibitor 2-benzylsuccinic acid, could efficiently inhibit CCP activities. 2-PMPA inhibited the recombinant Nna1 (a.k.a. CCP1) for hydrolyzing a synthetic peptide in a mixed manner, with Ki and Ki' being 0.11 µM and 0.24 µM respectively. It inhibited Nna1 for deglutamylating tubulin, the best-known polyglutamylated protein, with an IC50 of 0.21 mM. Homology modeling predicted that the R-form of 2-PMPA is more favorable to bind Nna1, unlike that GCPII prefers to S-form. This work for the first time identified a potent inhibitor for CCP family.

MRI Assessment of Prostate-Specific Membrane Antigen (PSMA) Targeting by a PSMA-Targeted Magnetic Nanoparticle: Potential for Image-Guided Therapy.

Magnetic nanoparticle (MNP)-induced hyperthermia is currently being evaluated for localized prostate cancer. We evaluated the feasibility of tumor-selective delivery of prostate-specific membrane antigen (PSMA)-targeted MNPs in a murine model with high-resolution magnetic resonance imaging (MRI) after intravenous administration of MNPs at a concentration necessary for hyperthermia. A PSMA-targeted MNP was synthesized and evaluated using T2-weighted MRI, after intravenous administration of 50 mg/kg of the MNP. Significant contrast enhancement ( P < 0.0002, n = 5) was observed in PSMA(+) tumors compared to PSMA(-) tumors 24 h and 48 h after contrast agent administration. Mice were also imaged with near-infrared fluorescence imaging, to validate the MRI results. Two-photon microscopy revealed higher vascular density at the tumor periphery, which resulted in higher  peripheral accumulation of PSMA-targeted MNPs. These results suggest that the delivery of PSMA-targeted MNPs to PSMA(+) tumors is both actively targeted and passively mediated.

The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity.

Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca2+ -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca2+ coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches. Our data unequivocally show that even a slight disruption of the Ca2+ -binding site destabilizes the three-dimensional fold of GCPII and is associated with impaired secretion, a high propensity to form nonphysiological oligomers, and an inability to bind active site-targeted ligands. Additionally, the Ca2+ -binding site is critical for maintenance of the native homodimeric quaternary arrangement of GCPII, which is indispensable for its enzymatic activity. Overall, our results offer a clear picture of the importance of Ca2+ for the structural integrity and hydrolytic activity of human GCPII and by extension homologous members of the M28 zinc-dependent metallopeptidase family.

Plumbagin-loaded aptamer-targeted poly D,L-lactic-co-glycolic acid-b-polyethylene glycol nanoparticles for prostate cancer therapy.

Plumbagin inhibits the growth, metastasis, and invasion of prostate cancer (PCa). However, its lower bioavailability limits biopharmaceutical properties due to insolubility in water. Prostate-specific membrane antigen (PSMA) aptamer-targeted nanoparticles (NPs) significantly enhanced cytotoxicity in prostate epithelial cells. This study aimed to investigate the effects of plumbagin-loaded prostate-specific membrane antigen (PSMA) aptamer-targeted poly D,L-lactic-co-glycolic acid-b-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) on prostate cancer (PCa) in vitro.PLGA-PEG with a terminal carboxylic acid group (PLGA-PEG-COOH) was synthesized, and plumbagin was loaded on PLGA-PEG-COOH NPs using the nanoprecipitation method and characterized by field emission scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser light scattering. The uptake and distribution of plumbagin-NPs in human PCa LNCaP cells were investigated by fluorescent labeling. Subsequently, PSMA antibody-targeted PLGA-PEG-COOH NPs (targeted NPs) were prepared by covalent binding and characterized by x-ray photoelectron spectroscopy. Furthermore, the anticancer activity of plumbagin-loaded, targeted NPs was compared with that of nontargeted NPs in LNCaP cells in vitro.Plumbagin-NPs (diameter of 189.4 ±â€Š30.6 nm and zeta potential of -17.1 ±â€Š3.7 mV) were optimized based on theoretical drug loading of 5% and a ratio of water:acetone of 3:1. During the first 2 hours, the cumulative release rate of the drug was 66.4 ±â€Š8.56%. Moreover, plumbagin-targeted NPs with nitrogen atoms were prepared. The uptake rate was 90% at 0.5 hours for targeted and nontargeted NPs. The IC50 of targeted NPs and nontargeted NPs was 32.59 ±â€Š8.03 µM and 39.02 ±â€Š7.64 µM, respectively.Plumbagin-loaded PSMA aptamer-targeted NPs can be used in targeted chemotherapy against PCa.

Optimization of Labeling PSMAHBED with Ethanol-Postprocessed 68Ga and Its Quality Control Systems.

Radiolabeling of the prostate-specific membrane antigen (PSMA) inhibitor Glu-NH-CO-NH-Lys(Ahx) using the 68Ga chelator HBED-CC (PSMAHBED) allows imaging of prostate cancer lesions because of high expression of PSMA in prostate carcinoma cells and in bone metastases and lymph nodes related to the disease. The aim of this work was to optimize labeling of 68Ga-PSMAHBED using the efficient cation-exchange postprocessing of 68Ga as well as the development of a thin-layer chromatography (TLC)-based quality control system. Methods: Labeling was optimized for online ethanol-postprocessed 68Ga eluate investigating various parameters, such as buffer molarity (0.1-1 M), temperature (25°C-90°C), tracer amount (0.11-0.74 nmol), and labeling time. In addition, purification of the crude product was tested. For radio-TLC quality control, various mobile phases were analyzed using silica gel 60 plates and the results were validated using high-performance liquid chromatography. The most superior mobile phases were also applied on instant thin-layer chromatography (ITLC) silica gel plates. Results: Using optimized conditions, labeling yields of more than 95% were obtained within 10 min when ethanol-based postprocessing was applied using PSMAHBED amounts as low as 0.1 nmol. A higher precursor concentration (0.7 nmol) further increased labeling and quantitative yields to more than 98% within 5 min. In clinical routine, patient batches (>200 applications) with radiochemical purity greater than 98% and specific activities of 326 ± 20 MBq/nmol are obtained reproducibly. When TLC quality control was performed on silica gel 60 plates, 4 mobile phases with suitable separation properties and complementary Rf values were identified. Two systems showed equivalent separation on ITLC silica gel plates, with ITLC analysis finished within 5 min, in contrast to 20 min for the TLC system. Labeling of PSMAHBED was optimized for cation-exchange postprocessing methods, ensuring almost quantitative labeling and high nuclide purity of final 68Ga-PSMAHBED, making subsequent purification steps unnecessary. Conclusion: The new radio-TLC method allows quality control in a short time using a fast, reliable, low-cost method with little equipment complexity. Using this approach, the synthesis is easily adopted by automated synthesis modules.

Development of a complementary PET/MR dual-modal imaging probe for targeting prostate-specific membrane antigen (PSMA).

We tried to develop a dual-modal PET/MR imaging probe using a straightforward one-pot method by encapsulation with specific amphiphiles. In this study, iron oxide (IO) nanoparticles were encapsulated with three amphiphiles containing PEG, DOTA and the prostate-specific membrane antigen (PSMA)-targeting ligand in aqueous medium. The diameter of the prepared nanoparticle DOTA-IO-GUL was 11.01±1.54nm. DOTA-IO-GUL was labeled with (68)Ga in high efficiency. The DOTA-IO-GUL showed a dose-dependent binding to LNCaP (PSMA positive) cells via a competitive binding study against (125)I-labeled MIP-1072 (PSMA-targeting agent). Additionally, PET and MR imaging results showed PSMA selective uptake by only 22Rv1 (PSMA positive) but not PC-3 (PSMA negative) in dual-tumor xenograft mouse model study. MR imaging showed high resolution, and PET imaging enabled quantification and confirmation of the specificity. In conclusion, we have successfully developed the specific PSMA-targeting IO nanoparticle, DOTA-IO-GUL, as a dual-modality probe for complementary PET/MR imaging. FROM THE CLINICAL EDITOR: The combination of using Positron Emission Tomography (PET) and computed tomography (CT) in clinical practice is now the norm. With advances in technology, the next step would be to develop combined PET and Magnetic Resonance (MR) dual-imaging. In this article, the authors described their positive study on the development of a dual-modal PET/MR imaging probe using a prostate cancer model.

Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II.

We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.

Crystal structure of prostate-specific membrane antigen, a tumor marker and peptidase.

Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer cells and nonprostatic solid tumor neovasculature and is a target for anticancer imaging and therapeutic agents. PSMA acts as a glutamate carboxypeptidase (GCPII) on small molecule substrates, including folate, the anticancer drug methotrexate, and the neuropeptide N-acetyl-l-aspartyl-l-glutamate. Here we present the 3.5-A crystal structure of the PSMA ectodomain, which reveals a homodimer with structural similarity to transferrin receptor, a receptor for iron-loaded transferrin that lacks protease activity. Unlike transferrin receptor, the protease domain of PSMA contains a binuclear zinc site, catalytic residues, and a proposed substrate-binding arginine patch. Elucidation of the PSMA structure combined with docking studies and a proposed catalytic mechanism provides insight into the recognition of inhibitors and the natural substrate N-acetyl-l-aspartyl-l-glutamate. The PSMA structure will facilitate development of chemotherapeutics, cancer-imaging agents, and agents for treatment of neurological disorders.