Jinbin Xu

Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumor cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor binding site. WC-21, a sigma-2 ligand containing both a photoactive moiety azide and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was then identified as PGRMC1 (progesterone receptor membrane component-1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalizes with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex.

Amyloid β Peptide–Induced Cerebral Endothelial Cell Death Involves Mitochondrial Dysfunction and Caspase Activation

Amyloid β peptide (Aβ), a 39 to 43 amino acid fragment of the β-amyloid precursor protein (βAPP), forms insoluble fibrillar accumulation in neurofibrillary tangles and vascular plaques. Aβ has been implicated in neuronal and vascular degeneration in brain regions susceptible to plaque formation because of its cytotoxic effect on neurons and endothelial cells (ECs). The authors used a murine cerebral endothelial cell (CEC) line and primary cultures of bovine CECs to explore the cytotoxic mechanism of Aβ. Aβ 1–40 and Aβ 25–35 peptides caused cell death in a dose-dependent and time-dependent manner. Exposure to either Aβ 25–35 or Aβ 1–40 at 10 μmol/L for 48 hours caused at least 40% cell death. Cerebral endothelial cell death was characterized by nuclear condensation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage. Aβ 25–35 activated both caspase-8 and caspase-3 in murine CECs. zVAD-fmk, a broad-spectrum caspase inhibitor, prevented Aβ 25–35-induced increase in caspase-3 activity and CEC death. N-acetyl-cysteine, an antioxidant, also prevented Aβ-induced cell death. Together, these findings indicate that Aβ-mediated CEC death is an apoptotic process that is characterized by increased oxidative stress, caspase activation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage.

Selective sigma-2 ligands preferentially bind to pancreatic adenocarcinomas: applications in diagnostic imaging and therapy

BackgroundResistance to modern adjuvant treatment is in part due to the failure of programmed cell death. Therefore the molecules that execute the apoptotic program are potential targets for the development of anti-cancer therapeutics. The sigma-2 receptor has been found to be over-expressed in some types of malignant tumors, and, recently, small molecule ligands to the sigma-2 receptor were found to induce cancer cell apoptosis.ResultsThe sigma-2 receptor was expressed at high levels in both human and murine pancreas cancer cell lines, with minimal or limited expression in normal tissues, including: brain, kidney, liver, lung, pancreas and spleen. Micro-PET imaging was used to demonstrate that the sigma-2 receptor was preferentially expressed in tumor as opposed to normal tissues in pancreas tumor allograft-bearing mice. Two structurally distinct sigma-2 receptor ligands, SV119 and WC26, were found to induce apoptosis to mice and human pancreatic cancer cells in vitro and in vivo. Sigma-2 receptor ligands induced apoptosis in a dose dependent fashion in all pancreatic cell lines tested. At the highest dose tested (10 μM), all sigma-2 receptor ligands induced 10–20% apoptosis in all pancreatic cancer cell lines tested (p < 0.05). In pancreas tumor allograft-bearing mice, a single bolus dose of WC26 caused approximately 50% apoptosis in the tumor compared to no appreciable apoptosis in tumor-bearing, vehicle-injected control animals (p < 0.0001). WC26 significantly slowed tumor growth after a 5 day treatment compared to vehicle-injected control animals (p < 0.0001) and blood chemistry panels suggested that there is minimal peripheral toxicity.ConclusionWe demonstrate a novel therapeutic strategy that induces a significant increase in pancreas cancer cell death. This strategy highlights a new potential target for the treatment of pancreas cancer, which has little in the way of effective treatments.

Subcellular Localization of Sigma-2 Receptors in Breast Cancer Cells Using Two-Photon and Confocal Microscopy

Sigma-2 receptor agonists have been shown to induce cell death via caspase-dependent and caspase-independent pathways. Unfortunately, there is little information regarding the molecular function of sigma-2 receptors that can explain these results. In this study, two fluorescent probes, SW107 and K05-138, were used to study the subcellular localization of sigma-2 receptors by two-photon and confocal microscopy. The results indicate that sigma-2 receptors colocalize with fluorescent markers of mitochondria, lysosomes, endoplasmic reticulum, and the plasma membrane in both EMT-6 mouse and MDA-MB-435 human breast cancer cells. The fluorescent probe, K05-138, was internalized rapidly, reaching a plateau of fluorescent intensity at 5 min. The internalization of K05-138 was reduced approximately 40% by phenylarsine oxide, an inhibitor of endocytosis. These data suggest that sigma-2 ligands are internalized, in part, by an endocytotic pathway. The localization of sigma-2 receptors in several organelles known to have a role in both caspase-dependent and caspase-independent pathways of cell death supports the conclusions of previous studies suggesting that sigma-2 receptor ligands should be evaluated as potential cancer chemotherapeutic agents.

The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine

BackgroundSigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy.ResultsThe binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities.ConclusionsThis study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.

Binding of the Radioligand SIL23 to α-Synuclein Fibrils in Parkinson Disease Brain Tissue Establishes Feasibility and Screening Approaches for Developing a Parkinson Disease Imaging Agent

Accumulation of α-synuclein (α-syn) fibrils in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson disease (PD). Ligands that bind α-syn fibrils could be utilized as imaging agents to improve the diagnosis of PD and to monitor disease progression. However, ligands for α-syn fibrils in PD brain tissue have not been previously identified and the feasibility of quantifying α-syn fibrils in brain tissue is unknown. We report the identification of the 125I-labeled α-syn radioligand SIL23. [125I]SIL23 binds α-syn fibrils in postmortem brain tissue from PD patients as well as an α-syn transgenic mouse model for PD. The density of SIL23 binding sites correlates with the level of fibrillar α-syn in PD brain tissue, and [125I]SIL23 binding site densities in brain tissue are sufficiently high to enable in vivo imaging with high affinity ligands. These results identify a SIL23 binding site on α-syn fibrils that is a feasible target for development of an α-syn imaging agent. The affinity of SIL23 for α-syn and its selectivity for α-syn versus Aβ and tau fibrils is not optimal for imaging fibrillar α-syn in vivo, but we show that SIL23 competitive binding assays can be used to screen additional ligands for suitable affinity and selectivity, which will accelerate the development of an α-syn imaging agent for PD.

Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain

2-((4-(1-[(11)C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)-quinoline (MP-10), a specific PDE10A inhibitor (IC(50)=0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by alkylation of the desmethyl precursor with [(11)C]CH(3)I, ∼45% yield, >92% radiochemical purity, >370 GBq/μmol specific activity at end of bombardment (EOB). Evaluation in Sprague-Dawley rats revealed that [(11)C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min striatum: cerebellum ratio reached 6.55. MicroPET studies of [(11)C]MP-10 in monkeys displayed selective uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood-brain-barrier may limit the clinical utility of [(11)C]MP-10 as a PDE10A PET tracer.

Synthesis and in Vitro and in Vivo Evaluation of 18F-Labeled Positron Emission Tomography (PET) Ligands for Imaging the Vesicular Acetylcholine Transporter

A new class of vesicular acetylcholine transporter inhibitor that incorporates a carbonyl group into the benzovesamicol structure was synthesized, and analogues were evaluated in vitro. (+/-)-trans-2-Hydroxy-3-(4-(4-[(18)F]fluorobenzoyl)piperidino)tetralin (9e) has K(i) values of 2.70 nM for VAChT, 191 nM for sigma(1), and 251 nM for sigma(2). The racemic precursor (9d) was resolved via chiral HPLC, and (+/-)-[(18)F]9e, (-)-[(18)F]9e, and (+)-[(18)F]9e were respectively radiolabeled via microwave irradiation of the appropriate precursors with [(18)F]/F(-) and Kryptofix/K(2)CO(3) in DMSO with radiochemical yields of approximately 50-60% and specific activities of >2000 mCi/micromol. (-)-[(18)F]9e uptake in rat brain was consistent with in vivo selectivity for the VAChT with an initial uptake of 0.911 %ID/g in rat striatum and a striatum/cerebellum ratio of 1.88 at 30 min postinjection (p.i.). MicroPET imaging of macaques demonstrated a 2.1 ratio of (-)-[(18)F]9e in putamen versus cerebellum at 2 h p.i. (-)-[(18)F]9e has potential to be a PET tracer for clinical imaging of the VAChT.

Dopamine D1, D2, D3 Receptors, Vesicular Monoamine Transporter Type-2 (VMAT2) and Dopamine Transporter (DAT) Densities in Aged Human Brain

The dopamine D1, D2, D3 receptors, vesicular monoamine transporter type-2 (VMAT2), and dopamine transporter (DAT) densities were measured in 11 aged human brains (aged 77–107.8, mean: 91 years) by quantitative autoradiography. The density of D1 receptors, VMAT2, and DAT was measured using [3H]SCH23390, [3H]dihydrotetrabenazine, and [3H]WIN35428, respectively. The density of D2 and D3 receptors was calculated using the D3-preferring radioligand, [3H]WC-10 and the D2-preferring radioligand [3H]raclopride using a mathematical model developed previously by our group. Dopamine D1, D2, and D3 receptors are extensively distributed throughout striatum; the highest density of D3 receptors occurred in the nucleus accumbens (NAc). The density of the DAT is 10–20-fold lower than that of VMAT2 in striatal regions. Dopamine D3 receptor density exceeded D2 receptor densities in extrastriatal regions, and thalamus contained a high level of D3 receptors with negligible D2 receptors. The density of dopamine D1 linearly correlated with D3 receptor density in the thalamus. The density of the DAT was negligible in the extrastriatal regions whereas the VMAT2 was expressed in moderate density. D3 receptor and VMAT2 densities were in similar level between the aged human and aged rhesus brain samples, whereas aged human brain samples had lower range of densities of D1 and D2 receptors and DAT compared with the aged rhesus monkey brain. The differential density of D3 and D2 receptors in human brain will be useful in the interpretation of PET imaging studies in human subjects with existing radiotracers, and assist in the validation of newer PET radiotracers having a higher selectivity for dopamine D2 or D3 receptors.

Carbon-11 labeled papaverine as a PET tracer for imaging PDE10A: radiosynthesis, in vitro and in vivo evaluation

Papaverine, 1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, a specific inhibitor of phosphodiesterase (PDE) 10A with IC(50) values of 36 nM for PDE10A, 1,300 nM for PDE3A and 320 nM for PDE4D, has served as a useful pharmaceutical tool to study the physiological role of PDE10A. Here, we report the radiosynthesis of [(11)C]papaverine and the in vitro and in vivo evaluation of [(11)C]papaverine as a potential positron emission tomography (PET) radiotracer for imaging PDE10A in the central nervous system (CNS). The radiosynthesis of papaverine with (11)C was achieved by O-methylation of the corresponding des-methyl precursor with [(11)C]methyl iodide. [(11)C]papaverine was obtained with approximately 70% radiochemical yield and a specific activity >10 Ci/mumol. In vitro autoradiography studies of rat and monkey brain sections revealed selective binding of [(11)C]papaverine to PDE10A enriched regions: the striatum of rat brain and the caudate and putamen of rhesus monkey brain. The biodistribution of [(11)C]papaverine in rats at 5 min demonstrated an initially higher accumulation in striatum than in other brain regions, however the washout was rapid. MicroPET imaging studies in rhesus macaques similarly displayed initial specific uptake in the striatum with very rapid clearance of [(11)C]papaverine from brain. Our initial evaluation suggests that despite papaverines utility for in vitro studies and as a pharmaceutical tool, [(11)C]papaverine is not an ideal radioligand for clinical imaging of PDE10A in the CNS. Analogs of papaverine having a higher potency for inhibiting PDE10A and improved pharmacokinetic properties will be necessary for imaging this enzyme with PET.