Development of an 18F-labeled azobenzothiazole tracer for α-synuclein aggregates in the brain.

Nuclear imaging of aggregated α-synuclein pathology is an urgent clinical need for Parkinson's disease, yet promising tracers for brain α-synuclein aggregates are still rare. In this work, a class of compact benzothiazole derivatives was synthesized and evaluated for α-synuclein aggregates. Among them, azobenzothiazoles exhibited specific and selective detection of α-synuclein aggregates under physiological conditions. Fluoro-pegylated azobenzothiazole NN-F further demonstrated high-affinity binding to α-synuclein aggregates and efficient 18F-radiolabeling via nucleophilic displacement of a tosyl precursor. [18F]NN-F was stable in plasma in vitro and showed efficient brain uptake with little defluorination in vivo.

Preclinical evaluation of 68Ga-radiolabeled trimeric affibody for PDGFRß-targeting PET imaging of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a highly vascularized solid carcinoma and tumor vessel-targeted molecular imaging might be effective for early diagnosis of HCC. Herein, we developed a novel trimeric affibody (ZTRI) with highly specific binding to the platelet-derived growth factor receptor beta (PDGFRß). The aim of this study is to evaluate the feasibility of 68Ga-radiolabeled ZTRI ([68Ga]Ga-DOTA-ZTRI) as PET tracer for diagnosis of HCC. METHODS: The bioinformatics analysis of clinical database and immunoblotting of clinical specimens were performed to validate the potential of PDGFRß as HCC biomarker. The trimeric affibody ZTRI was conjugated with DOTA-NHS-ester and radiolabeled with 68Ga to produce [68Ga]Ga-DOTA-ZTRI conjugate. Immunoreactivity and specific uptake of [68Ga]Ga-DOTA-ZTRI were assessed by dose-dependent cell binding, autoradiography, and biodistribution analysis. [68Ga]Ga-DOTA-ZTRI PET/CT scanning of diethylnitrosamine (DEN)-induced primary HCC rats and a rare case of idiopathical HCC rhesus monkey was performed to evaluate the imaging capability and radiation dosimetry of [68Ga]Ga-DOTA-ZTRI in vivo. RESULTS: Excessive PDGFRß was validated as a representative biomarker of HCC neovascularization. The radiolabeling of [68Ga]Ga-DOTA-ZTRI was achieved at more than 95% radiochemical yield. In vitro assays showed specific uptake of [68Ga]Ga-DOTA-ZTRI in HCC tumor vessels by autoradiography. Animal PET/CT imaging with [68Ga]Ga-DOTA-ZTRI successfully visualized the tumor lesions in primary HCC rats and rhesus monkey, and indicated radiation absorbed dose of 2.03E-02 mSv/MBq for each scanning. CONCLUSIONS: Our results demonstrated that [68Ga]Ga-DOTA-ZTRI conjugate could be applied as a promising PET tracer for early diagnosis of hepatocellular carcinoma.

Structure of the full-length glucagon class B G-protein-coupled receptor.

The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a ß-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a ß-hairpin conformation and interacts with the stalk to form a compact ß-sheet structure. Hydrogen-deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.

Synthesis, radiolabeling, and evaluation of a potent ß-site APP cleaving enzyme (BACE1) inhibitor for PET imaging of BACE1 in vivo.

The ß-site APP-cleaving enzyme 1 (BACE1) plays important roles in the proteolytic processing of amyloid precursor protein, and can be regarded as an important target for the diagnosis and treatment of AD. This study aimed to report the synthesis and evaluation of an 18F-labeled 2-amino-3,4-dihydroquinazoline analog as a potential BACE1 radioligand. A fluoropropyl side chain was introduced to the phenyl of this 3,4-dihydroquinazoline scaffold to generate the radioligand. Our preliminary data indicated that although the 2-amino-3,4-dihydroquinazoline scaffold possessed favorable in-vitro properties as a PET ligand, its poor brain uptake hindered the in-vivo imaging of BACE1. Further investigation would be required to optimize the scaffold for the development of a blood-brain-barrier-permeable BACE1-targeted PET ligand.

64Cu-labeled melanin nanoparticles for PET/CT and radionuclide therapy of tumor.

Melanin is a group of natural pigments found in living organism. It can be used for positron emission tomography (PET) imaging due to its inherent chelating ability to radioactive cupric ion. This study was to prepare 64Cu-labeled PEGylated melanin nanoparticles (64Cu-PEG-MNPs), and to further take advantage of the enhanced permeability and retention (EPR) effect of radiolabeled nanoparticles to realize the integration of tumor diagnosis and treatment. We successfully synthesized PEG-MNPs. Saline and serum stability experiments demonstrated good stability. PET/CT showed high tumor aggregation. Moreover, 64Cu-PEG-MNPs resulted in a therapeutic effect on the A431 tumor-bearing mice in the treatment group. The pathological results further confirmed that the therapeutic doses of 64Cu-PEG-MNPs cause pathological changes of tumor tissues while showing minimal toxicity to normal tissues. Our data successfully demonstrate the good imaging performance of 64Cu-PEG-MNPs on A431 tumors and further proved its therapeutic effect, highlighting a great potential in targeted radionuclide therapy.

[Recent progress of positron radionuclide labeled small molecule inhibitors of prostate specific membrane antigen for PET/CT imaging of prostate cancer].

Prostate cancer is the most common tumor of the urinary system, and its mortality rate is second only to lung cancer. With the specific and high expression on the surface of prostate cancer cells, prostate-specific membrane antigen (PSMA) has been an ideal theranostic target of prostate cancer with great clinical significance and research value. Positron emission tomography/computed tomography (PET/CT), a new modality of molecular imaging combining functional metabolic information and anatomical structure, provides high diagnostic performance for cancer detection. This paper mainly reviewed recent progress of PSMA inhibitors labeled by positron-emitting radionuclides for early diagnosis, preoperative staging, response assessment, restaging and metastasis detection of prostate cancer.

Positron Emission Tomography Imaging of Platelet-Derived Growth Factor Receptor ß in Colorectal Tumor Xenograft Using Zirconium-89 Labeled Dimeric Affibody Molecule.

Platelet-derived growth factor receptor ß (PDGFRß) is overexpressed in a variety of malignant cancers, plays a critical role in tumor angiogenesis, and has been proven as a valuable target for cancer treatment. In this pilot study, a dimeric affibody molecule, ZPDGFRß, was prepared and radiolabeled with positron emission radionuclide zirconium-89 for PET imaging of colorectal tumors by targeting PDGFRß expression in vivo. The PDGFRß-binding capability of dimeric affibody was evaluated by flow cytometry, immunofluorescent staining, and whole-body optical imaging. Then, ZPDGFRß was conjugated with DFO-Bn-NCS and radiolabeled with 89Zr. Targeted binding capability of 89Zr-DFO-ZPDGFRß to PDGFRß expressing cells was investigated by cellular assay in vitro and microPET/CT imaging in vivo. Dimeric ZPDGFRß affibody had specifically higher binding capability with PDGFRß expressing pericytes rather than LS-174T cancer cells, and well colocalized with tumor neovasculature by flow cytometry and immunofluorescent assay. ZPDGFRß was successfully labeled with 89Zr by DFO chelating with yield of 94.1 ± 3.53%. 89Zr-DFO-ZPDGFRß indicated preserved specific binding ability with PDGFRß expressing cells and effective inhibiting capability to PDGF-ß ligands ( P < 0.05) in vitro. Biodistribution indicated that tumor uptake of 89Zr-DFO-ZPDGFRß reached the peak of 6.93 ± 0.64%ID/g, and the tumor-to-blood ratio was 5.5 ± 0.6 at 2 h post-injection. LS-174T xenografts were clearly visualized by microPET/CT imaging through 1 to 4 h post-injection of 89Zr-DFO-ZPDGFRß affibody conjugate. In conclusion, the 89Zr-DFO-ZPDGFRß conjugate demonstrated specific and high binding ability with colorectal tumor, which indicated its use as a potential radiopharmaceutical for diagnostic imaging of tumor associate vasculatures with PET/CT.

Organocatalytic and Scalable Syntheses of Unsymmetrical 1,2,4,5-Tetrazines by Thiol-Containing Promotors.

Despite the growing application of tetrazine bioorthogonal chemistry, it is still challenging to access tetrazines conveniently from easily available materials. Described here is the de novo formation of tetrazine from nitriles and hydrazine hydrate using a broad array of thiol-containing catalysts, including peptides. Using this facile methodology, the syntheses of 14 unsymmetric tetrazines, containing a range of reactive functional groups, on the gram scale were achieved with satisfactory yields. Using tetrazine methylphosphonate as a building block, a highly efficient Horner-Wadsworth-Emmons reaction was developed for further derivatization under mild reaction conditions. Tetrazine probes with diverse functions can be scalably produced in yields of 87-93 %. This methodology may facilitate the widespread application of tetrazine bioorthogonal chemistry.

Alkoxy Tetrazine Substitution at a Boron Center: A Strategy for Synthesizing Highly Fluorogenic Hydrophilic Probes.

Strongly fluorogenic boron dipyrromethene (BODIPY)-tetrazine probes have been obtained by introducing an alkoxy tetrazine fragment at the boron center. The fluorescence signal from these probes strongly increases by up to 225-fold after reaction with bioorthogonal coupling partners, and the hydrophilicity of probes is improved, such that they are suitable for live-cell imaging.

Novel Function of Extracellular Matrix Protein 1 in Suppressing Th17 Cell Development in Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS characterized by demyelination and axonal damage. Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model for human MS. Although Th17 cells are important for disease induction, Th2 cells are inhibitory in this process. In this article, we report the effect of a Th2 cell product, extracellular matrix protein 1 (ECM1), on the differentiation of Th17 cells and the development of EAE. Our results demonstrated that ECM1 administration from day 1 to day 7 following the EAE induction could ameliorate the Th17 cell responses and EAE development in vivo. Further study of the mechanism revealed that ECM1 could interact with αv integrin on dendritic cells and block the αv integrin-mediated activation of latent TGF-ß, resulting in an inhibition of Th17 cell differentiation at an early stage of EAE induction. Furthermore, overexpression of ECM1 in vivo significantly inhibited the Th17 cell response and EAE induction in ECM1 transgenic mice. Overall, our work has identified a novel function of ECM1 in inhibiting Th17 cell differentiation in the EAE model, suggesting that ECM1 may have the potential to be used in clinical applications for understanding the pathogenesis of MS and its diagnosis.

Targeted Imaging of Tumor-Associated Macrophages by Cyanine 7-Labeled Mannose in Xenograft Tumors.

Mannose receptor is considered as a hallmark of M2-oriented tumor-associated macrophages (TAMs), but its utility in TAMs was rarely reported. Therefore, deoxymannose (DM), a high-affinity ligand of mannose receptor, was labeled with near-infrared dye cyanine 7 (Cy7), and its feasibility of targeted imaging on TAMs was evaluated in vitro and in vivo. The Cy7-DM was synthesized, and its binding affinity with induced TAMs in vitro, whole-body imaging in xenograft tumor mouse model in vivo, and the cellular localization in dissected tissues were evaluated. We demonstrated a high uptake of Cy7-DM by induced M2 macrophages and TAMs in tumor tissues. In vivo near-infrared live imaging visualized abundant TAMs in tumor lesions instead of inflammatory sites by Cy7-DM imaging, and the quantity of Cy7-DM signals in tumors was significantly higher than that shown in inflammatory sites from 1 to 8 hours of imaging. Our results suggest that mannose could rapidly and specifically target TAMs and is a promising candidate for targeted diagnosis of tumor with rich TAMs.

Discovery of pyrazolo[1,5-a]pyrimidine-3-carbonitrile derivatives as a new class of histone lysine demethylase 4D (KDM4D) inhibitors.

Herein we report the discovery of a series of new small molecule inhibitors of histone lysine demethylase 4D (KDM4D). Molecular docking was first performed to screen for new KDM4D inhibitors from various chemical databases. Two hit compounds were retrieved. Further structural optimization and structure-activity relationship (SAR) analysis were carried out to the more selective one, compound 2, which led to the discovery of several new KDM4D inhibitors. Among them, compound 10r is the most potent one with an IC50 value of 0.41±0.03µM against KDM4D. Overall, compound 10r could be taken as a good lead compound for further studies.

[Primary study on fluro [ 19F] berberine derivative for human hepatocellular carcinoma targetting in vitro].

[ 18F]HX-01, a Fluorine-18 labeled berberine derivative, is a potential positron emission tomography (PET) tumor imaging agent, while [ 19F]HX-01 is a nonradioactive reference substance with different energy state and has the same physical and chemical properties. In order to collect data for further study of [ 18F]HX-01 PET imaging of hepatocellular carcinoma in vivo, this study compared the uptake of [ 19F]HX-01 by human hepatocellular carcinoma and normal hepatocytes in vitro. The target compound, [ 19F]HX-01, was synthesized in one step using berberrubine and 3-fluoropropyl 4-methylbenzenesulfonate. Cellular uptake and localization of [ 19F]HX-01 were performed by a fluorescence microscope in human hepatocellular carcinoma HepG2, SMMC-7721 and human normal hepatocyte HL-7702. Cellular proliferation inhibition and cell cytotoxicity assay of the [ 19F]HX-01 were conducted using cell counting kit-8 (CCK-8) on HepG2, SMMC-7721 and HL-7702 cells. Fluorescent microscopy showed that the combining ability of [ 19F]HX-01 to the carcinoma SMMC-7721 and HepG2 was higher than that to the normal HL-7702. Cellular proliferation inhibition assay demonstrated that [ 19F]HX-01 leaded to a dose-dependent inhibition on SMMC-7721, HepG2, and HL-7702 proliferation. Cell cytotoxicity assay presented that the cytotoxicity of [ 19F]HX-01 to SMMC-7721 and HepG2 was obviously higher than that to HL-7702. This in vitro study showed that [ 19F]HX-01 had a higher selectivity on human hepatocellular carcinoma cells (SMMC-7721, HepG2) but has less toxicity to normal hepatocytes (HL-7702). This could set up the idea that the radioactive reference substance [ 18F]HX-01 may be worthy of further development as a potential molecular probe targeting human hepatocellular carcinoma using PET.

Discovery of KDM5A inhibitors: Homology modeling, virtual screening and structure-activity relationship analysis.

Herein we report the discovery of a series of new KDM5A inhibitors. A three-dimensional (3D) structure model of KDM5A jumonji domain was firstly established based on homology modeling. Molecular docking-based virtual screening was then performed against commercial chemical databases. A number of hit compounds were retrieved. Further structural optimization and structure-activity relationship (SAR) analysis were carried out to the most active hit compound, 9 (IC50: 2.3 µM), which led to the discovery of several new KDM5A inhibitors. Among them, compound 15e is the most potent one with an IC50 value of 0.22 µM against KDM5A. This compound showed good selectivity for KDM5A and considerable ability to suppress the demethylation of H3K4me3 in intact cells. Compound 15e could be taken as a good lead compound for further studies.

[Cellular Uptake and Localization of Novel NSCLC Penetrating Peptide with Neuropilin-1 Binding Motif].

OBJECTIVE: To synthesize and study the specific binding affinity of tumor-penetrating peptide YCCS to non-small cell lung carcinoma (NSCLC) cells in vitro. METHODS: YCCS peptide was designed by fusing the neuropilin-1 (NRP-1) binding sequence and NSCLC binding peptide CS. YCCS peptide was synthesized and fluorescent labeled with N-terminal FITC. NRP-1 positive human NSCLC cell A549, NRP-1 positive human breast cancer cell MDA-MB-231, normal human bronchial epithelium HBE135-E6E7 and human liver cell HL-7702 were incubated respectively, then we observed the specific binding affinity of tumor-penetrating peptide YCCS to NSCLC cells. RESULTS: After treated with 5 µmol/L peptide, significant fluorescent signals of FITC-YCCS peptide were demonstrated only in NSCLC A549 cells but marginal captured signal in MDA-MB-231, normal human HBE135-E6E7 or HL-7702 cells, which revealed specific NSCLC cells binding affinity. In 20 µmol/L treated group, non-specific binding were found in MDA-MB-231 and HL-7702 cells. CONCLUSION: The results of this novel designed YCCS peptide indicated a promising strategy for improving tumor penetrating with delivery capability of drugs to NSCLC A549 cells when treated with 5 µmol/L peptide.

Discovery and structure-activity analysis of 4-((5-nitropyrimidin-4-yl)amino)benzimidamide derivatives as novel protein arginine methyltransferase 1 (PRMT1) inhibitors.

Despite a potential application of PRMT1 inhibitors in cancer treatment, very few of PRMT1 inhibitors have been reported. To obtain novel potent PRMT1 inhibitors, structure optimizations towards a hit compound, 4-((6-chloro-5-nitropyrimidin-4-yl)amino)benzimidamide, were carried out. A series of 4-((5-nitropyrimidin-4-yl)amino)benzimidamide derivatives were synthesized. Structure-activity relationship analysis led to the discovery of a number of PRMT1 inhibitors. The most potent compound corresponds to compound 6d, which showed an IC50 value of 2.0 µM against PRMT1. This compound also displayed a considerable anti-proliferative activity against three tumor cell lines, DLD-1, T24 and SH-SY-5Y, with IC50 values of 4.4 µM, 13.1 µM and 11.4 µM, respectively.

[18F-Berberine Derivatives: a Potential Molecular Imaging Agent for Tumor Targeting by PET/CT Tumor].

Cancer is one of the main causes of death for human beings. Clinical oncologists increasingly rely upon imaging for diagnosis, stage, response assessment, and follow-up in cancer patient. However, 18F-FDG is not a tumor specific agent, inflammation and infection also have intensive uptake of 18F-FDG, resulting in false positive diagnosis, and some tumors have low uptake of 18F-FDG or even do not uptake 18F-FDG, leading to false negative diagnosis. So it is urgent to develop non-18F-FDG novel tumor targeting agent. Recently, a large number of researches in vitro have demonstrated that berberine has anti-tumor activity against a variety of tumor cells by inducing tumor cell apoptosis through inhibition of mitochondrial respiratory chain etc. So far, there is no credible evidence of berberine targeting in tumor in vivo. We proposed a hypothesis that berberine has the characteristics of tumor targeting biodistribution in vivo, and verified the proposal by 18F-berberine PET/CT imaging in VX2 muscle tumor-bearing rabbit model. In this review, we intend to give an overview of the progress of berberine anticancer, the structural bases of berberine anticancer and the uderlying molecular mechanisms of berberine anticancer indentified so far. We also introduce the first visualization of 18F labeled berberine derivatives targeting tumor in VX2 muscle tumor-bearing rabbit model by PET/CT. These breakthrough findings suggest that 18F-berberine derivatives as a potential PET/CT tumor targeted molecular imaging agent may have important implications for cancer targeting therapy, molecular imaging and modernization of Traditional Chinese Medicine.

Recent progress of imaging agents for Parkinson's disease.

Parkinson's disease (PD) is a common progressive, neurodegenerative brain disease that is promoted by mitochondrial dysfunction, oxidative stress, protein aggregation and proteasome dysfunction in the brain. Compared with computer tomography (CT) or magnetic resonance imaging (MRI), non-invasive nuclear radiopharmaceuticals have great significance for the early diagnosis of PD due to their high sensitivity and specificity in atypical and preclinical cases. Based on the development of coordination chemistry and chelator design, radionuclides may be delivered to lesions by attaching to PD-related transporters and receptors, such as dopamine, serotonin, and others. In this review, we comprehensively detailed the current achievements in radionuclide imaging in Parkinson's disease.

X-ray Structure-Guided Discovery of a Potent Benzimidazole Glutaminyl Cyclase Inhibitor That Shows Activity in a Parkinson's Disease Mouse Model.

The secretory glutaminyl cyclase (sQC) and Golgi-resident glutaminyl cyclase (gQC) are responsible for N-terminal protein pyroglutamation and associated with various human diseases. Although several sQC/gQC inhibitors have been reported, only one inhibitor, PQ912, is currently undergoing clinic trials for the treatment of Alzheimer's disease. We report an X-ray crystal structure of sQC complexed with PQ912, revealing that the benzimidazole makes "anchor" interactions with the active site zinc ion and catalytic triad. Structure-guided design and optimization led to a series of new benzimidazole derivatives exhibiting nanomolar inhibition for both sQC and gQC. In a MPTP-induced Parkinson's disease (PD) mouse model, BI-43 manifested efficacy in mitigating locomotor deficits through reversing dopaminergic neuronal loss, reducing microglia, and decreasing levels of the sQC/gQC substrates, α-synuclein, and CCL2. This study not only offers structural basis and new leads for drug discovery targeting sQC/gQC but also provides evidence supporting sQC/gQC as potential targets for PD treatment.

Transcriptome Analysis Reveals Candidate Genes Involved in Gibberellin-Induced Fruit Development in Rosa roxburghii.

Gibberellins (GAs) play indispensable roles in the fruit development of horticultural plants. Unfortunately, the molecular basis behind GAs regulating fruit development in R. roxburghii remains obscure. Here, GA3 spraying to R. roxburghii 'Guinong 5' at full-bloom promoted fruit size and weight, prickle development, seed abortion, ascorbic acid accumulation, and reduction in total soluble sugar. RNA-Seq analysis was conducted to generate 45.75 Gb clean reads from GA3- and non-treated fruits at 120 days after pollination. We obtained 4275 unigenes belonging to differently expressed genes (DEGs). Gene ontology and the Kyoto Encyclopedia of Genes and Genomes displayed that carbon metabolism and oxidative phosphorylation were highly enriched. The increased critical genes of DEGs related to pentose phosphate, glycolysis/gluconeogenesis, and citrate cycle pathways might be essential for soluble sugar degradation. Analysis of DEGs implicated in ascorbate revealed the myoinositol pathway required to accumulate ascorbic acid. Finally, DEGs involved in endogenous phytohormones and transcription factors, including R2R3 MYB, bHLH, and WRKY, were determined. These findings indicated that GA3-trigged morphological alterations might be related to the primary metabolites, hormone signaling, and transcription factors, providing potential candidate genes that could be guided to enhance the fruit development of R. roxburghii in practical approaches.