Computational insights into the aggregation mechanism and amyloidogenic core of aortic amyloid medin polypeptide.

Medin amyloid, prevalent in the vessel walls of 97 % of individuals over 50, contributes to arterial stiffening and cerebrovascular dysfunction, yet our understanding of its aggregation mechanism remains limited. Dividing the full-length 50-amino-acid medin peptide into five 10-residue segments, we conducted individual investigations on each segment's self-assembly dynamics via microsecond-timescale atomistic discrete molecular dynamics (DMD) simulations. Our findings showed that medin1-10 and medin11-20 segments predominantly existed as isolated unstructured monomers, unable to form stable oligomers. Medin31-40 exhibited moderate aggregation, forming dynamic ß-sheet oligomers with frequent association and dissociation. Conversely, medin21-30 and medin41-50 segments demonstrated significant self-assembly capability, readily forming stable ß-sheet-rich oligomers. Residue pairwise contact frequency analysis highlighted the critical roles of residues 22-26 and 43-49 in driving the self-assembly of medin21-30 and medin41-50, acting as the ß-sheet core and facilitating ß-strand formation in other regions within medin monomers, expecting to extend to oligomers and fibrils. Regions containing residues 22-26 and 43-49, with substantial self-assembly abilities and assistance in ß-sheet formation, represent crucial targets for amyloid inhibitor drug design against aortic medial amyloidosis (AMA). In summary, our study not only offers deep insights into the mechanism of medin amyloid formation but also provides crucial theoretical and practical guidance for future treatments of AMA.

Enhanced Prostate-specific Membrane Antigen Targeting by Precision Control of DNA Scaffolded Nanoparticle Ligand Presentation.

Targeted nanoparticles have been extensively explored for their ability to deliver their payload to a selective cell population while reducing off-target side effects. The design of actively targeted nanoparticles requires the grafting of a ligand that specifically binds to a highly expressed receptor on the surface of the targeted cell population. Optimizing the interactions between the targeting ligand and the receptor can maximize the cellular uptake of the nanoparticles and subsequently improve their activity. Here, we evaluated how the density and presentation of the targeting ligands dictate the cellular uptake of nanoparticles. To do so, we used a DNA-scaffolded PLGA nanoparticle system to achieve efficient and tunable ligand conjugation. A prostate-specific membrane antigen (PSMA) expressing a prostate cancer cell line was used as a model. The density and presentation of PSMA targeting ligand ACUPA were precisely tuned on the DNA-scaffolded nanoparticle surface, and their impact on cellular uptake was evaluated. It was found that matching the ligand density with the cell receptor density achieved the maximum cellular uptake and specificity. Furthermore, DNA hybridization-mediated targeting chain rigidity of the DNA-scaffolded nanoparticle offered ∼3 times higher cellular uptake compared to the ACUPA-terminated PLGA nanoparticle. Our findings also indicated a ∼ 3.7-fold reduction in the cellular uptake for the DNA hybridization of the non-targeting chain. We showed that nanoparticle uptake is energy-dependent and follows a clathrin-mediated pathway. Finally, we validated the preferential tumor targeting of the nanoparticles in a bilateral tumor xenograft model. Our results provide a rational guideline for designing actively targeted nanoparticles and highlight the application of DNA-scaffolded nanoparticles as an efficient active targeting platform.

Covalent targeted radioligands potentiate radionuclide therapy.

Targeted radionuclide therapy, in which radiopharmaceuticals deliver potent radionuclides to tumours for localized irradiation, has addressed unmet clinical needs and improved outcomes for patients with cancer1-4. A therapeutic radiopharmaceutical must achieve both sustainable tumour targeting and fast clearance from healthy tissue, which remains a major challenge5,6. A targeted ligation strategy that selectively fixes the radiopharmaceutical to the target protein in the tumour would be an ideal solution. Here we installed a sulfur (VI) fluoride exchange (SuFEx) chemistry-based linker on radiopharmaceuticals to prevent excessively fast tumour clearance. When the engineered radiopharmaceutical binds to the tumour-specific protein, the system undergoes a binding-to-ligation transition and readily conjugates to the tyrosine residues through the 'click' SuFEx reaction. The application of this strategy to a fibroblast activation protein (FAP) inhibitor (FAPI) triggered more than 80% covalent binding to the protein and almost no dissociation for six days. In mice, SuFEx-engineered FAPI showed 257% greater tumour uptake than did the original FAPI, and increased tumour retention by 13-fold. The uptake in healthy tissues was rapidly cleared. In a pilot imaging study, this strategy identified more tumour lesions in patients with cancer than did other methods. SuFEx-engineered FAPI also successfully achieved targeted ß- and α-radionuclide therapy, causing nearly complete tumour regression in mice. Another SuFEx-engineered radioligand that targets prostate-specific membrane antigen (PSMA) also showed enhanced therapeutic efficacy. Considering the broad scope of proteins that can potentially be ligated to SuFEx warheads, it might be possible to adapt this strategy to other cancer targets.

PSMA-targeted SMART molecules outfitted with SN38.

Herein, we report the modular synthesis and evaluation of a prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugate (SMDC) carrying the chemotherapeutic agent, SN38. Due to the fluorogenic properties of SN38, payload release kinetics from the platform was observed in buffers representing the pH conditions of systemic circulation and cellular internalization. It was found that this platform is stable with minimal payload release at physiological pH with most rapid payload release observed at pH values representing the endosome complex. We confirmed selective payload release and chemotherapeutic efficacy for PSMA(+) prostate cancer cells over PSMA(-) cells. These results demonstrate that chemotherapeutic agents with limited solubility can be conjugated to a water-soluble targeting and linker platform without attenuating efficacy.

Autoradiographic characterization of [18 F]PSMA-1007 binding in rat brain.

Carboxypeptidase II (CBPII) in brain metabolizes the neuroactive substance N-acetyl-L-aspartyl-L-glutamate (NAGG) to yield the elements of glutamate and N-acetyl-aspartate (NAA). In peripheral organs, CBPII is known as prostrate specific membrane antigen (PSMA), which presents an important target for nuclear medicine imaging in prostate cancer. Available PSMA ligands for PET imaging do not cross the blood-brain barrier, and there is scant knowledge of the neurobiology of CBPII, despite its implication in the regulation of glutamatergic neurotransmission. In this study we used the clinical PET tracer [18 F]-PSMA-1007 ([18 F]PSMA) for an autoradiographic characterization of CGPII in rat brain. Ligand binding and displacement curves indicated a single site in brain, with KD of about 0.5 nM, and Bmax ranging from 9 nM in cortex to 19 nM in white matter (corpus callosum and fimbria) and 24 nM in hypothalamus. The binding properties of [18 F]PSMA in vitro should enable its use for autoradiographic investigations of CBPII expression in animal models of human neuropsychiatric conditions.

Identification of alternative protein targets of glutamate-ureido-lysine associated with PSMA tracer uptake in prostate cancer cells.

Prostate-specific membrane antigen (PSMA) is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-specific probes incorporating glutamate-ureido-lysine (GUL). PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based PSMA tracers are still reported to have the potential to identify NEPC metastatic tumors. These probes may bind unknown proteins associated with PSMA-suppressed cancers. We have identified the up-regulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in PSMA-suppressed prostate cancers and find that their expression levels inversely correlate with PSMA expression and are associated with GUL-based radiotracer uptake. Furthermore, we identify that NAALADaseL and mGluR expression correlates with a unique cell cycle signature. This provides an opportunity for the future study of the biology of NEPC and potential therapeutic directions. Computationally predicting that GUL-based probes bind well to these targets, we designed and synthesized a fluorescent PSMA tracer to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL, and specific mGluRs associated with poor prognosis.

The utility of radiolabeled PSMA ligands for tumor imaging.

Prostate-specific membrane antigen (PSMA) is a glycosylated type-II transmembrane protein expressed in prostatic tissue and significantly overexpressed in several prostate cancer cells. Despite its name, PSMA has also been reported to be overexpressed in endothelial cells of benign and malignant non-prostate disease. So its clinical use was extended to detection, staging, and therapy of various tumor types. Recently small molecules targeting PSMA have been developed as imaging probes for diagnosis of several malignancies. Preliminary studies are emerging improved diagnostic sensitivity and specificity of PSMA imaging, leading to a change in patient management. In this review, we evaluated the first preclinical and clinical studies on PSMA ligands resulting future perspectives radiolabeled PSMA in staging and molecular characterization, based on histopathologic examinations of PSMA expression.

Synthesis, Characterization, and Preclinical Evaluation of a Small-Molecule Prostate-Specific Membrane Antigen-Targeted Monomethyl Auristatin E Conjugate.

Prostate cancer is the second most common type of cancer among men. Its main method of treatment is chemotherapy, which has a wide range of side effects. One of the solutions to this challenge is targeted delivery to prostate cancer cells. Here we synthesized a novel small-molecule PSMA-targeted conjugate based on the monomethyl auristatin E. Its structure and conformational properties were investigated by NMR spectroscopy. Cytotoxicity, intracellular reactive oxygen species induction, and stability under liver microsomes and P450-cytochrome species were investigated for this conjugate. The conjugate demonstrated 77-85% tumor growth inhibition levels on 22Rv1 (PSMA (+)) xenografts, compared with a 37% inhibition level on PC-3 (PSMA (-)) xenografts, in a single dose of 0.3 mg/kg and a sufficiently high therapeutic index of 21. Acute, chronic, and subchronic toxicities and pharmacokinetics have shown that the synthesized conjugate is a promising potential agent for the chemotherapy of prostate cancer.

Hybrid Chelator-Based PSMA Radiopharmaceuticals: Translational Approach.

(1) Background: Prostate-specific membrane antigen (PSMA) has been extensively studied in the last decade. It became a promising biological target in the diagnosis and therapy of PSMA-expressing cancer diseases. Although there are several radiolabeled PSMA inhibitors available, the search for new compounds with improved pharmacokinetic properties and simplified synthesis is still ongoing. In this study, we developed PSMA ligands with two different hybrid chelators and a modified linker. Both compounds have displayed a promising pharmacokinetic profile. (2) Methods: DATA5m.SA.KuE and AAZTA5.SA.KuE were synthesized. DATA5m.SA.KuE was labeled with gallium-68 and radiochemical yields of various amounts of precursor at different temperatures were determined. Complex stability in phosphate-buffered saline (PBS) and human serum (HS) was examined at 37 °C. Binding affinity and internalization ratio were determined in in vitro assays using PSMA-positive LNCaP cells. Tumor accumulation and biodistribution were evaluated in vivo and ex vivo using an LNCaP Balb/c nude mouse model. All experiments were conducted with PSMA-11 as reference. (3) Results: DATA5m.SA.KuE was synthesized successfully. AAZTA5.SA.KuE was synthesized and labeled according to the literature. Radiolabeling of DATA5m.SA.KuE with gallium-68 was performed in ammonium acetate buffer (1 M, pH 5.5). High radiochemical yields (>98%) were obtained with 5 nmol at 70 °C, 15 nmol at 50 °C, and 60 nmol (50 µg) at room temperature. [68Ga]Ga-DATA5m.SA.KuE was stable in human serum as well as in PBS after 120 min. PSMA binding affinities of AAZTA5.SA.KuE and DATA5m.SA.KuE were in the nanomolar range. PSMA-specific internalization ratio was comparable to PSMA-11. In vivo and ex vivo studies of [177Lu]Lu-AAZTA5.SA.KuE, [44Sc]Sc-AAZTA5.SA.KuE and [68Ga]Ga-DATA5m.SA.KuE displayed specific accumulation in the tumor along with fast clearance and reduced off-target uptake. (4) Conclusions: Both KuE-conjugates showed promising properties especially in vivo allowing for translational theranostic use.

A prostate-specific membrane antigen activated molecular rotor for real-time fluorescence imaging.

Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.

Rapid and specific detection nanoplatform of serum exosomes for prostate cancer diagnosis.

Tumor exosomes that inherit specific molecules from their parent cells are emerging as ideal biomarkers in cancer diagnostics. Most currently available exosome isolation and detection methods are time-consuming and non-specific; thus, rapid and specific exosome detection methods are needed both clinically and in research. Here, a dual-functional platform is reported composed of reversible conjunction and "off-on" signal responses. Fe3O4@SiO2@TiO2 particles with high affinity were applied to capture exosomes, and model exosomes could be isolated from solution within 20 min with a capture efficiency of 91.5%. An "on-off" fluorescence response PSMA aptasensor was constructed with improved selectivity to detect tumor exosomes by recording the fluorescence intensity with λex/em = 557/580 nm. The standard curve for detecting tumor exosomes with the aptasensor was calculated as y = 371.7x + 66.17, ranging from 0.05 to 1 × 104 particles/µL, with R2 = 0.9737, and a detection limit of 5 × 102 particles/µL in solution. This method was successfully applied to clinical samples, and the results showed better performance in distinguishing prostate cancer patients and healthy samples than the traditional nanoparticle-tracking analysis (NTA) method. This rapid and accurate detection method for prostate cancer may aid in rapid clinical diagnosis. Integrating quickly TiO2-based isolation with sensitive and specific "on-off" detection of PCa exosomes.

Synthesis and in vitro preliminary evaluation of prostate-specific membrane antigen targeted upconversion nanoparticles as a first step towards radio/fluorescence-guided surgery of prostate cancer.

Over the last decade, upconversion nanoparticles (UCNP) have been widely investigated in nanomedicine due to their high potential as imaging agents in the near-infrared (NIR) optical window of biological tissues. Here, we successfully develop active targeted UCNP as potential probes for dual NIR-NIR fluorescence and radioactive-guided surgery of prostate-specific membrane antigen (PSMA)(+) prostate cancers. We designed a one-pot thermolysis synthesis method to obtain oleic acid-coated spherical NaYF4:Yb,Tm@NaYF4 core/shell UCNP with narrow particle size distribution (30.0 ± 0.1 nm, as estimated by SAXS analysis) and efficient upconversion luminescence. Polyethylene glycol (PEG) ligands bearing different anchoring groups (phosphate, bis- and tetra-phosphonate-based) were synthesized and used to hydrophilize the UCNP. DLS studies led to the selection of a tetra-phosphonate PEG(2000) ligand affording water-dispersible UCNP with sustained colloidal stability in several aqueous media. PSMA-targeting ligands (i.e., glutamate-urea-lysine derivatives called KuEs) and fluorescent or radiolabelled prosthetic groups were grafted onto the UCNP surface by strain-promoted azide-alkyne cycloaddition (SPAAC). These UCNP, coated with 10 or 100% surface density of KuE ligands, did not induce cytotoxicity over 24 h incubation in LNCaP-Luc or PC3-Luc prostate cancer cell lines or in human fibroblasts for any of the concentrations evaluated. Competitive binding assays and flow cytometry demonstrated the excellent affinity of UCNP@KuE for PSMA-positive LNCaP-Luc cells compared with non-targeted UCNP@CO2H. Furthermore, the binding of UCNP@KuE to prostate tumour cells was positively correlated with the surface density of PSMA-targeting ligands and maintained after 125I-radiolabelling. Finally, a preliminary biodistribution study in LNCaP-Luc-bearing mice demonstrated the radiochemical stability of non-targeted [125I]UCNP paving the way for future in vivo assessments.

PSMA-targeted low-molecular double conjugates for diagnostics and therapy.

This review presents data on dual conjugates of therapeutic and diagnostic action for targeted delivery to prostate cancer cells. The works of the last ten years on this topic were analyzed. The mail attention focuses on low-molecular-weight conjugates directed to the prostate-specific membrane antigen (PSMA); the comparison of high and low molecular weight PSMA-targeted conjugates was made. The considered conjugates were divided in the review into two main classes: diagnostic bimodal conjugates (which are containing two fragments for different types of diagnostics), theranostic conjugates (containing both therapeutic and diagnostic agents); also bimodal high molecular weight therapeutic conjugates containing two therapeutic agents are briefly discussed. The data of in vitro and in vivo studies for PSMA-targeted double conjugates available by the beginning of 2021 have been analyzed.

Automatic radiosynthesis and preclinical evaluation of 18F-AlF-PSMA-NF as a potential PET probe for prostate cancer imaging.

Facile automatic production is important for the application of prostate-specific membrane antigen (PSMA) tracers in clinical practice. We developed a new 18F-AlF-labelled PSMA probe-18F-AlF-PSMA-NF-and explore its automated production method and potential value in clinical settings. 18F-AlF-PSMA-NF was prepared using an automated method with dimethylformamide (DMF) as the solvent in a positron emission tomography (PET)-MF-2 V-IT-I synthesizer. Tracer characteristics were examined both in vitro and in vivo. Micro-PET/computed tomography (CT) was performed to investigate the utility of 18F-AlF-PSMA-NF for imaging PSMA-positive tumours in vivo. 18F-AlF-PSMA-NF was prepared automatically within 35 min with a non-attenuation correction yield of 37.9 ± 11.2%. The tracer was hydrophilic, had a high affinity for PSMA (Kd = 2.58 ± 0.81 nM), and showed stability in both in vitro and in vivo conditions. In the cellular experiments, 18F-AlF-PSMA-NF uptake in PSMA-positive LNCaP cells was significantly higher than that in PSMA-negative PC-3 cells (P < 0.001), and could be blocked by excess ZJ-43-a PSMA inhibitor (P < 0.001). LNCaP tumours were clearly visualized by 18F-AlF-PSMA-NF on micro-PET/CT, with a high level of uptake (13.72 ± 2.01 percent injected dose per gram of tissue [%ID/g]) and high tumour/muscle ratio (close to 50:1). The PSMA-positive LNCaP tumours had a significantly higher uptake than PSMA-negative PC-3 tumours (13.72 ± 2.01%ID/g vs. 1.07 ± 0.48%ID/g, t = 10.382, P < 0.001), and could be blocked by ZJ-43 (13.72 ± 2.01%ID/g vs. 2.77 ± 1.44%ID/g, t = 8.14, P < 0.001). A new 18F-AlF-labelled PSMA probe-18F-AlF-PSMA-NF-was successfully developed and can be prepared automatically. It has the biological characteristics resembling that of a PSMA-based probe and can potentially be used in clinical settings.

Radiolabeling Optimization and Preclinical Evaluation of the New PSMA Imaging Agent [18F]AlF-P16-093.

Prostate-specific membrane antigen (PSMA)-targeted radioligands have played an increasing role in the diagnosis of prostate cancer. [68Ga]Ga-P16-093 is a PSMA-targeting agent for positron emission tomography imaging, currently under a Phase 2 clinical trial. In the present study, P16-093 was labeled with 18F via [18F]AlF2+ complex formation, and the biological properties of [18F]AlF-P16-093 were evaluated. Optimization of radiolabeling efficiency was performed by testing a series of parameters, including the amount of free ligand; the amount of Al3+; and the influence of solvent, pH, temperature, reaction time, and reaction volume. Optimal labeling results were achieved at pH 5 by reacting at 60 °C for 15 min in a vial containing 74-370 MBq of [18F]fluoride, 46 nmol of P16-093, 40 nmol of AlCl3·6 H2O, and 50% EtOH. [18F]AlF-P16-093 was prepared with a non-decay-corrected radiochemical yield of 54.4 ± 4.4% (n = 9) within 30 min (final radiochemical purity ≥95%). In vitro, [18F]AlF-P16-093 showed PSMA-specific high uptakes in PIP-PC3 cells. The binding affinity of [18F]AlF-P16-093 to PSMA was determined as Kd of 12.4 ± 2.0 nM. The tumor uptake in mice with a xenografted PSMA-expressing PIP-PC3 tumor was high (18.8 ± 5.14% ID/g at 1 h postinjection) and retained without washout for 2 h. In addition, tumor uptake was almost completely blocked by coinjecting a PSMA inhibitor, 2-PMPA. The bone activity at 1 h post injection was higher with [18F]AlF-P16-093 (2.83 ± 0.49% ID/g) in comparison to that of [68Ga]Ga-P16-093 (0.26 ± 0.07% ID/g). In summary, an efficient and simple radiosynthesis of [18F]AlF-P16-093 was achieved. [18F]AlF-P16-093 showed desirable in vivo pharmacokinetics and excellent PSMA-targeting properties for imaging PSMA expression in prostate cancer.

A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.

Synthesis and Biological Evaluation of PSMA Ligands with Aromatic Residues and Fluorescent Conjugates Based on Them.

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is a suitable target for specific delivery of antitumor drugs and diagnostic agents due to its overexpression in prostate cancer cells. In the current work, we describe the design, synthesis, and biological evaluation of novel low-molecular PSMA ligands and conjugates with fluorescent dyes FAM-5, SulfoCy5, and SulfoCy7. In vitro evaluation of synthesized PSMA ligands on the activity of PSMA shows that the addition of aromatic amino acids into a linker structure leads to a significant increase in inhibition. The conjugates of the most potent ligand with FAM-5 as well as SulfoCy5 demonstrated high affinities to PSMA-expressing tumor cells in vitro. In vivo biodistribution in 22Rv1 xenografts in Balb/c nude mice of PSMA-SulfoCy5 and PSMA-SulfoCy7 conjugates with a novel PSMA ligand demonstrated good visualization of PSMA-expressing tumors. Also, the conjugate PSMA-SulfoCy7 demonstrated the absence of any explicit toxicity up to 87.9 mg/kg.

A Structure-Activity Relationship Study of Bimodal BODIPY-Labeled PSMA-Targeting Bioconjugates.

The aim of this study was to identify a high-affinity BODIPY peptidomimetic that targets the prostate-specific membrane antigen (PSMA) as a potential bimodal imaging probe for prostate cancer. For the structure-activity study, several BODIPY (difluoroboron dipyrromethene) derivatives with varying spacers between the BODIPY dye and the PSMA Glu-CO-Lys binding motif were prepared. Corresponding affinities were determined by competitive binding assays in PSMA-positive LNCaP cells. One compound was identified with comparable affinity (IC50 =21.5±0.1 nM) to Glu-CO-Lys-Ahx-HBED-CC (PSMA-11) (IC50 =18.4±0.2 nM). Radiolabeling was achieved by Lewis-acid-mediated 19 F/18 F exchange in moderate molar activities (∼0.7 MBq nmol-1 ) and high radiochemical purities (>99 %) with mean radiochemical yields of 20-30 %. Cell internalization of the 18 F-labeled high-affinity conjugate was demonstrated in LNCaP cells showing gradual increasing PSMA-mediated internalization over time. By fluorescence microscopy, localization of the high-affinity BODIPY-PSMA conjugate was found in the cell membrane at early time points and also in subcellular compartments at later time points. In summary, a high-affinity BODIPY-PSMA conjugate has been identified as a suitable candidate for the development of PSMA-specific dual-imaging agents.

Simple new method for labelling of PSMA-11 with 68Ga in NaHCO3.

BACKGROUND: Prostate specific membrane antigen (PSMA) is a type II membrane protein widely expressed on the surface of prostate cancer cells. One of its functions is to act as a receptor mediating the ligand internalization. This PSMA property is employed in the diagnostics and therapy of prostate cancer. Over the years, small molecules with high affinity for PSMA have been developed and labelled with positron emitters (e.g. 68Ga, 18F, 11C, 64Cu, or 86Y). One of these radiolabelled ligands, [68Ga] PSMA-11, is one of the most widespread tracers for PET imaging of the prostate cancer. Many techniques have been proposed and tested for the 68Ga labelling of PSMA-11. The aim of our work was to design a labelling method of PSMA-11 that minimizes number of the used chemicals and steps, providing quantitative labelling yield at laboratory temperature and may be easily automated. METHODOLOGY: A68Ge/68Ga generator eluate in 0.1 M HCl was loaded on an activated Oasis MCX cartridge, and the cartridge was then thoroughly washed with water. The radionuclide 68Ga was eluted from the cartridge with 0.1 M NaHCO3 (pH = 8.5, n = 36) or with the same solution with pH adjusted to 7.2-9.0 (n = 38). Precursor PSMA-11 was mixed directly with the cartridge eluate of 68Ga in 0.1 M NaHCO3 of given pH. For the stability test, samples of 68GaPSMA-11 in 0.1 M NaHCO3 (pH 8.5) were mixed in ratio 1 : 1 with the following solutions: 0.1 M NaHCO3 (pH 8.5), human serum, PBS and 0.9% NaCl. In order to estimate an effect of the time elapsed between 68Ga elution from the cartridge in 0.1 M NaHCO3 (pH 8.5) and the labelling onset of PSMA-11, the latter was initiated 0, 5, 10 and 20 min post elution and radiochemical yield was monitored. All the PSMA-11 labelled samples were subjected to radiochemical purity test using HPLC. The whole process starting from generator elution up to HPLC analysis commencement took 10-15 min. RESULTS: Recovery of 68Ga from cartridge Oasis MCX using 0.1 M NaHCO3 at pH 8.5 was 71.5 ± 1.4%. Thirty six PSMA-11 samples (10 µg in reaction mixture) were labelled at pH 8.5 with total average radiochemical yield of 98 ± 2%. Recovery of 68Ga from cartridge Oasis MCX using 0.1 M NaHCO3 at variable pH of 7.2-9.0 was 62.5 ± 1.8% showing certain decrease with decreasing pH. A total of 138 samples of PSMA-11 were labelled with 68 Ga at variable pH (7.2-9.0) and four different amounts of PSMA-11 (1, 2.5, 5 and 10 µg) resulting in the labelling yields of 54.0 ± 5.3%, 88.2 ± 3.2%, 99.4 ± 0.3% and 99.9 ± 0.1%, respectively. Irrespective of the pH, the radiolabelling yield was quantitative for the molar ratio PSMA-11: 68Ga > 5000 : 1 in the reaction mixture. Stability tests in 0.1 M NaHCO3 (pH 8.5), human serum, PBS and 0.9% NaCl revealed no observable release of 68Ga from the 68Ga-PSMA-11 complex within 3 h. Similarly, the delay between the 68Ga elution from the Oasis MCX cartridge in 0.1 M NaHCO3 (pH 8.5) and start of the labelling of PSMA-11 labelling has no effect on the radiochemical yield. CONCLUSION: A new method of labelling PSMA-11 ligand with 68Ga in 0.1 M NaHCO3 using Oasis MCX cartridges was proposed, developed and tested. The results demonstrated that it is rapid, simple, reproducible and easy to automate.

Potential effective inhibitory compounds against Prostate Specific Membrane Antigen (PSMA): A molecular docking and molecular dynamics study.

One of the most prevalent cancers in men is prostate cancer and could be managed with immunotoxins or antibody treatment. Because of the substantial rise of the Prostate-Specific Antigen and the Prostate-Specific Membrane Antigen (PSMA), cancer vaccination should be rendered with these antigens. Through pharmacodynamic experiments in a library of natural compounds from ZINC database, the current research sought to identify compounds that could suppress PSMA protein. To test the most productive compounds for further research, the Library has been scanned with Pharmacophore and ADMET analysis followed by molecular docking methods in the first phase. After selecting 15 ligands with the best pose related to docking results, to evaluate the stability of the ligand-protein bounds of the compounds, a molecular dynamics simulation considering the effect of the presence of zinc ions on the protein structure was performed. The measurement of ligand binding modes and free energy has shown that four compounds, including Z10, Z06, Z01, and Z03, have formed critical interactions with the active site's residues. Besides, multiple approaches were employed to determine their inhibition rating and describe the variables that facilitate the attachment of ligands to the protein active site. The results are obtained from the MMPBSA/GBSA analysis of four selected small molecules (Z10, Z06, Z01, and Z03), which are very close to the IC50 value of reference ligand (DCIBzl); they are -13.85 kcal/mol, -12.58 kcal/mol, -10.71 kcal/mol and -9.39 kcal/mol respectively. Finally, we evaluate the results obtained from selected ligands using hydrogen bond and decomposition analyzes. We have examined the effective interactions between ligands and S1/S1'pockets in protein. Our computational results illustrate the design of more efficient inhibitors of PSMA.