The sigma-2 (σ2) receptor is an important target for the development of molecular probes in oncology because of its 10-fold higher density in proliferating tumor cells than in quiescent tumor cells, and the observation that σ2 receptor agonists are able to kill tumor cells via apoptotic and non-apoptotic mechanisms. Although recent evidence indicates the σ2 receptor binding site is localized within the progesterone receptor membrane component 1 (PGRMC1), most information regarding this protein has been obtained using either radiolabeled or fluorescent receptor-based probes, and from biochemical analysis of the effect of σ2 selective ligands on cells grown in culture. This article reviews the development of σ2 receptor ligands, and presents an overview of how they have been used in vitro and in vivo to increase our understanding of the role of the σ2 receptor in cancer and proliferation.
Sigma-2 receptors; cell proliferation; radiotracer; fluorescent probe; positron emission tomography
A series of N-(2-methoxyphenyl)homopiperazine analogs was prepared and their affinities for dopamine D2, D3, and D4 receptors were measured using competitive radioligand binding assays. Several ligands exhibited high binding affinity and selectivity for the D3 dopamine receptor compared to the D2 receptor subtype. Compounds 11a, 11b, 11c, 11f, 11j and 11k had Ki values ranging from 0.7–3.9 nM for the D3 receptor with 30- to 170-fold selectivity for the D3 vs. D2 receptor. Calculated log P values (log P = 2.6–3.6) are within the desired range for passive transport across the blood brain barrier. When the binding and the intrinsic efficacy of these phenylhomopiperazines was compared to those of previously published phenylpiperazine analogues, it was found that a) affinity at D2 and D3 dopamine receptors generally decreased, b) the D3 receptor binding selectivity (D2:D3
Ki value ratio) decreased and, c) the intrinsic efficacy, measured using a forskolin-dependent adenylyl cyclase inhibition assay, generally increased.
Dopamine D2-like receptors; D3 dopamine receptors; receptor subtype selective ligands; homopiperazine analogs
A series of microPET imaging studies were conducted in anesthetized rhesus monkeys using the dopamine D2-selective partial agonist, [11C]SV-III-130. There was a high uptake in regions of brain known to express a high density of D2 receptors under baseline conditions. Rapid displacement in the caudate and putamen, but not in the cerebellum, was observed after injection of the dopamine D2/3 receptor nonselective ligand S(−)-eticlopride at a low dosage (0.025 mg/kg/i.v.); no obvious displacement in the caudate, putamen and cerebellum was observed after the treatment with a dopamine D3 receptor selective ligand WC-34 (0.1 mg/kg/i.v.). Pretreatment with lorazepam (1 mg/kg, i.v. 30 min) to reduce endogenous dopamine prior to tracer injection resulted in unchanged binding potential (BP) values, a measure of D2 receptor binding in vivo, in the caudate and putamen. D-amphetamine challenge studies indicate that there is a significant displacement of [11C]SV-III-130 by d-amphetamine-induced increases in synaptic dopamine levels.
Dopamine; Dopamine D2 receptors; Positron Emission Tomography
Two α-synuclein ligands, 3-methoxy-7-nitro-10H-phenothiazine (2a, Ki = 32.1 ± 1.3 nM) and 3-(2-fluoroethoxy)-7-nitro-10H-phenothiazine (2b, Ki = 49.0 ± 4.9 nM), were radiolabeled as potential PET imaging agents by respectively introducing 11C and 18F. The syntheses of [11C]2a and [18F]2b were accomplished in a good yield with high specific activity. Ex vivo biodistribution studies in rats revealed that both [11C]2a and [18F]2b crossed the blood-brain barrier (BBB) and demonstrated good brain uptake 5 min post-injection. MicroPET imaging of [11C]2a in a non-human primate (NHP) confirmed that the tracer was able to cross the BBB with rapid washout kinetics from brain regions of a healthy macaque. The initial studies suggested that further structural optimization of [11C]2a and [18F]2b is necessary in order to identify a highly specific positron emission tomography (PET) radioligand for in vivo imaging of α-synuclein aggregation in the central nervous system (CNS).
Lewy bodies; Parkinson's disease; PET; phenothiazine; radiosynthesis; α-synuclein
Traumatic events generate some of the most enduring forms of memories. Despite the elevated lifetime prevalence of anxiety disorders, effective strategies to attenuate long-term traumatic memories are scarce. The most efficacious treatments to diminish recent (i.e., day-old) traumata capitalize on memory updating mechanisms during reconsolidation that are initiated upon memory recall. Here, we show that, in mice, successful reconsolidation-updating paradigms for recent memories fail to attenuate remote (i.e., month-old) ones. We find that, whereas recent memory recall induces a limited period of hippocampal neuroplasticity mediated, in part, by S-nitrosylation of HDAC2 and histone acetylation, such plasticity is absent for remote memories. However, by using an HDAC2-targeting inhibitor (HDACi) during reconsolidation, even remote memories can be persistently attenuated. This intervention epigenetically primes the expression of neuroplasticity-related genes, which is accompanied by higher metabolic, synaptic, and structural plasticity. Thus, applying HDACis during memory reconsolidation might constitute a treatment option for remote traumata.
Pathogenic autosomal recessive mutations in the DJ-1 (Park7) or the PTEN-induced putative kinase 1 (Pink1 or PARK6) genes are associated with familial Parkinson’s disease (PD). It is not well known regarding the pathological mechanisms involving the DJ-1 and Pink1 mutations. Here we characterized DJ-1 and Pink1 knockout rats both through expression profiling and using quantitative autoradiography to measure the densities of the dopamine D1, D2, D3 receptors, vesicular monoamine transporter type-2 (VMAT2) and dopamine transporter (DAT) in the striatum of transgenic rats and wild type controls. Expression profiling with a commercially available array of 84 genes known to be involved in PD indicated that only the target gene was significantly downregulated in each transgenic rat model. D1 receptor, VMAT2, and DAT were measured using [3H]SCH23390, [3H]dihydrotetrabenazine, and [3H]WIN35428, respectively. No significant changes were observed in the density of DAT in either model. Although the densities of VMAT2 and D1 receptor were unchanged in Pink1 knockout, but both were increased in DJ-1 knockout rats. The densities of D2 and D3 receptors, determined by mathematical analysis of binding of radioligands [3H]WC-10 and [3H]raclopride, were significantly increased in both knockout models. These distinctive changes in the expression of dopamine presynaptic markers and receptors in the striatum may reflect different compensatory regulation of dopamine system in DJ-1 versus Pink1 knockout rat models of familial PD.
DJ-1; Pink1; Parkinson’s disease; dopamine receptors; autoradiography
WC 44 and WC 10 are phenylpiperazines with low (23 fold) to moderate (42 fold) selectivity for dopamine D3 receptors (D3Rs) over D2Rs, respectively. WC 44 is a full D3R agonist in the forskolin-stimulated adenylyl cyclase (AC) assay, whereas WC 10 has little efficacy. In contrast to their opposite effects in the AC assay, these drugs often produce similar behavioral effects, suggesting that the AC assay does not predict the efficacy of these drugs in vivo. Here we examined whether Fos protein expression induced by these drugs would be more consistent with their behavioral effects in vivo. Rats received either vehicle, WC 10 (5.6 mg/kg, i.p.), WC 44 (10.0 mg/kg, i.p), cocaine (10.0 mg/kg, i.p.), or cocaine with WC 10 (5.6 mg/kg, i.p.) or with WC 44 (10.0 mg/kg, i.p). Locomotion was monitored for 90 min and the brains were harvested for immunohistochemistry. Both WC 10 and WC 44 decreased spontaneous and cocaine-induced locomotion. Both compounds also increased Fos expression relative to saline in the dorsal striatum and nucleus accumbens core and shell, and relative to cocaine alone in the nucleus accumbens shell. The findings suggest that even though these compounds have different efficacy in the AC bioassy, they produce similar brain activation and attenuation of cocaine hyperlocomotion. Together with our previous research demonstrating that these compounds down-shift the cocaine self-administration dose-effect function, the findings support the idea that D3R-selective compounds may be useful for cocaine dependence medications development.
c-fos; immediate early gene; striatum; nucleus accumbens
Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.
Click labeling using 2-[18F]fluoroethyl azide has been proven to be promising methods of radiolabeling small molecules and peptides, some of which are undergoing clinical evaluations. However, the previously reported method afforded low yield, poor purities and under desirable reproducibility.
A vacuum distillation method was used to isolate 2-[18F]fluoroethyl azide, and the solvent effect of acetonitrile (ACN) and dimethylformamide (DMF) on the click labeling using Cu(I) from copper sulfate/sodium ascorbate was studied. The labeling conditions were optimized to radiosynthesize a hydroxysuccinimide ester (NHS).
2-[18F]fluoroethyl azide was isolated by the vacuum distillation method with > 80% yield within 10 min in a “pure” and click-ready form. It was found that the amount of DMF was critical for maintaining high levels of Cu(I) from copper sulfate/sodium ascorbate in order to rapidly complete the click labeling reaction. The addition of bathophenanthrolinedisulfonic acid disodium salt (BPDS) to the mixture of copper sulfate/sodium ascorbate also greatly improved the click labeling efficiency. Through exploiting these optimizations, a base-labile N-hydroxysuccinimide (NHS) ester was rapidly radiosynthesized in 90% isolated yield with good chemical and radiochemical purities.
We have developed a general method to click-label small molecules efficiently using [18F]2 for research and clinical use. This NHS ester can be used for conjugation chemistry to label antibodies, peptides and small molecules as PET tracers.
PET; Fluorine-18; fluoroethyl azide; click chemistry; hydroxysuccinimide ester
The regulation of D3 receptor has not been well documented in diffuse Lewy body disease (DLBD). In this study, a novel D3 preferring radioligand [3H]WC-10 and a D2-preferring radioligand [3H]raclopride were used and the absolute densities of the dopamine D3 and D2 receptors were determined in the striatal regions and substantia nigra (SN) from postmortem brains from 5 cases DLBD, which included dementia with Lewy bodies (DLB, n=4) and Parkinson disease dementia (PDD, n=1). The densities of the dopamine D1 receptor, vesicular monoamine transporter 2(VMAT2), and dopamine transporter (DAT) were also measured by quantitative autoradiography using [3H]SCH23390, [3H]dihydrotetrabenazine, and [3H]WIN35428, respectively. The densities of these dopaminergic markers were also measured in the same brain regions in 10 age-matched control cases. Dopamine D3 receptor density was significantly increased in the striatal regions including caudate, putamen and nucleus accumbens (NAc). There were no significant changes in the dopamine D1 and D2 receptor densities in any brain regions measured. VMAT2 and DAT densities were reduced in all the brain regions measured in DLB/PDD, however the significant reduction was found in putamen for DAT and in the NAc and SN for VMAT2. The decrease of dopamine pre-synaptic markers implies neuronal loss in the substantia nigra pars compacta (SNpc) in these DLB/PDD cases, while the increase of D3 receptors in striatal regions could be attributed to dopaminergic medication history and psychiatric state such as hallucinations. Whether it also reflects compensatory regulation upon dopaminergic denervation warrants further confirmations on larger populations.
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.
A panel of novel D2 and D3 dopamine receptor selective antagonists, partial agonists and full agonists have been evaluated for the ability to attenuate L-dopa associated abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA) unilaterally lesioned male Sprague Dawley rats, which is an animal model of L-dopa-induced dyskinesia (LID). LID is often observed in patients with Parkinson’s Disease following chronic treatment with L-dopa. The intrinsic activity of these dopaminergic compounds was determined using a forskolin-dependent adenylyl cyclase inhibition assay with transfected HEK 293 cells expressing either the human D2Long or D3 dopamine receptor subtype. For the initial experiments the 5-HT1A receptor selective partial agonist buspirone was used to verify our ability to quantitate changes in total AIMs and AIMs minus locomotor scores. Two D2 dopamine receptor selective antagonists, SV156 and SV293, were evaluated and found to minimally attenuate AIM scores in these animals. Four members of our WC series of D3 dopamine receptor selective compounds of varying intrinsic activity at the D3 dopamine receptor subtype, WC 10, WC 21, WC 26 and WC 44, were also evaluated and found to attenuate AIM scores in a dose dependent manner. The in vivo efficacy of the compounds increased when they were administered simultaneously with L-dopa, as compared to when the compounds were administered 60 minutes prior to the L-dopa/benserazide. It was also found that the D3 receptor antagonist WC 10 could inhibit the involuntary movements after they had achieved maximum intensity. Unlike the D1-like dopamine receptor selective agonist SKF 81297 and the D2-like dopamine receptor agonist bromocriptine which can precipitate abnormal involuntary movements in these unilaterally lesioned animals, abnormal involuntary movements were not observed after administration of our D3 receptor selective agonist WC 44. In addition, we evaluated the effect of these four D3 dopamine receptor selective compounds for their effect on a) spontaneous locomotion and b) coordination and agility using a rotarod apparatus. We also used a cylinder test to assess the effect of L-dopa on spontaneous and independent use of each of the rat’s forelimbs in the presence or absence of test compound. The results of these studies suggest that substituted phenylpiperazine D3 dopamine receptor selective compounds are potential pharmacotherapeutic agents for the treatment of L-dopa-associated dyskinesia in patients with Parkinson’s Disease.
Parkinson’s Disease; dyskinesia; L-dopa; dopamine receptors; D3 dopamine receptors
Peroxisome proliferator activated-receptor gamma (PPARγ) transcriptionally modulates fat metabolism and also plays a role in pathological conditions such as cancer, neurodegenerative disease, and inflammation. PPARγ imaging agents are potential tools for investigating these diseases.
Four analogs of GW9662, a PPARγ antagonist, with different fluorine-containing substituents at the para-position of the aniline ring were synthesized and evaluated using two different receptor binding assays for measuring PPARγ affinity. MicroPET imaging studies were performed in a transgenic mouse model having a heart specific overexpression of PPARγ.
All four analogs were found to have binding affinities that were comparable to or better than the reference antagonist, GW9662, using a scintillation proximity assay. However, only the chloro-based analogs (compounds 3 and 4) had activity in a whole cell assay measuring activation of the PPARγ / RXR receptor complex. MicroPET imaging studies in a MHC-PPARγ transgenic mouse model showed high uptake and PPARγ specific binding for the irreversible antagonist [18F]3, whereas the corresponding reversible methoxy analog ([18F]5) displayed only nonspecific uptake in heart.
The results of this preliminary study show that the irreversible antagonist [18F]3 may represent a novel strategy for imaging PPARγ in vivo with PET. 1.
PPARγ; peroxisome proliferator-activated receptor-γ; antagonist; imaging agent
We previously reported that the antipsychotic drug haloperidol, a multifunctional D2-like dopamine and sigma receptor subtype antagonist, has neuroprotective properties. In this study we further examined the association between neuroprotection and receptor antagonism by evaluating a panel of novel compounds with varying affinity at sigma and D2-like dopamine receptors. These compounds were evaluated using an in vitro cytotoxicity assay that utilizes a hippocampal-derived cell line, HT-22, in the presence or absence of varying concentrations (5 to 20 mM) of glutamate. While haloperidol was found to be a potent neuroprotective agent in this in vitro cell assay, the prototypic sigma 1 receptor agonist (+)-pentazocine was found not to be neuroprotective. Subsequently, the potency for the neuroprotection of HT-22 cells was evaluated for a) three SV series indoles which have nMolar affinity at D2-like receptors but varying affinity at sigma 1 receptor and b) two benzyl phenylacetamides sigma 1 receptor selective compounds which bind with low affinity at D2-like receptors but have nMolar affinity for the sigma 1 receptor. We observed that cytoprotection correlated with the affinity of the compounds for sigma 1 receptors. Based upon results from the HT-22 cell-based in vitro assay, two phenylacetamides, LS-127 and LS-137, were further evaluated in vivo using a transient middle cerebral artery occlusion (t-MCAO) model of stroke. At a dose of 100 µg/kg, both LS-127 and LS-137 attenuated infarct volume by approximately 50%. These studies provide further evidence that sigma 1 receptor selective compounds can provide neuroprotection in cytotoxic situations. These results also demonstrate that sigma 1 receptor selective benzyl phenylacetamides are candidate pharmacotherapeutic agents that could be used to minimize neuronal death after a stroke or head trauma.
Sigma receptors; Sigma 1 receptors; Neuroprotection
Subtype selective dopamine receptor ligands have long been sought after as therapeutic and/or imaging agents for the treatment and monitoring of neurologic disorders. We report herein on a combined structure- and ligand-based approach to explore the molecular mechanism of the subtype selectivity for a large class of D2-like dopamine receptor ligands (163 ligands in total). Homology models were built for both human D2l and D3 receptors in complex with haloperidol. Other ligands, which included multiple examples of substituted phenylpiperazines, were aligned against the binding conformations of haloperidol and three dimensional quantitative structure activity relationship (3D-QSAR) analyses were carried out. The receptor models show that although D2 and D3 share highly similar folds and 3D conformations, the slight sequence differences at their extracellular loop regions result in the binding cavity in D2 being comparably shallower than in D3, which may explain why some larger ligands bind with greater affinity at D3 compared to D2 receptors. The QSAR models show excellent correlation and high predictive power even when evaluated by the most stringent criteria. They confirm that the origins of subtype selectivity for the ligands arise primarily due to differences in the contours of the two binding sites. The predictive models suggest that while both steric and electrostatic interactions contribute to the compounds’ binding affinity, the major contribution arises from hydrophobic interactions, with hydrogen bonding conferring binding specificity. The current work provides clues for the development of more subtype selective dopamine receptor ligands. Furthermore, it demonstrates the possibility of being able to apply similar modeling methods to other subtypes or classes of receptors to study GPCR receptor-ligand interactions at a molecular level.
Dopamine receptor; subtype selective; homology; molecular dynamics; CoMFA
A series of microPET imaging studies were conducted in anesthetized rhesus monkeys using the dopamine D3-selective partial agonist, [18F]5. There was variable uptake in regions of brain known to express a high density of D3 receptors under baseline conditions. Pretreatment with lorazepam (1 mg/kg, i.v. 30 min) to reduce endogenous dopamine activity prior to tracer injection resulted in a dramatic increase in uptake in the caudate, putamen, and thalamus, and an increase in the binding potential (BP) values, a measure of D3 receptor binding in vivo. These data indicate that there is a high level of competition between [18F]5 and endogenous dopamine for D3 receptors in vivo.
D3 receptors; Positron Emission Tomography; endogenous dopamine
One major barrier in the development of pancreas cancer therapeutics is the selective delivery of the drugs to their cellular targets. We have developed previously several sigma-2 ligands and reported the discovery of a component of the receptor for these ligands. Several sigma-2 ligands have been shown to trigger apoptosis in pancreas cancer cells. More importantly sigma-2 ligands are internalized rapidly by the cancer cells, and are capable of delivering other small molecule therapeutics. Here we review sigma-2 ligands and conjugates as a potential novel therapy suitable for investigation in patients with pancreatic cancer.
Four benzamide analogs having a high affinity and selectivity for D3 versus D2 receptors were radiolabeled with 11C or 18F for in vivo evaluation.
Precursors were synthesized and the four D3 selective benzamide analogs were radiolabeled. The tissue distribution and brain uptake of the four compounds were evaluated in control rats and rats pretreated with cyclosporin A, a modulator of P-glycoprotein and an inhibitor of other ABC efflux transporters that contribute to the blood brain barrier. MicroPET imaging was carried out for [11C]6 in a control and a cyclosporin A pre-treated rat.
All four compounds showed low brain uptake in control rats at 5 and 30 min post-injection; despite recently reported rat behavioral studies conducted on analogs 6 (WC-10) and 7 (WC-44). Following administration of cyclosporin A, increased brain uptake was observed with all four PET radiotracers at both 5 and 30 min post-i.v. injection. An increase in brain uptake following modulation/inhibition of the ABC transporters was also observed in the microPET study.
These data suggest that D3 selective conformationally-flexible benzamide analogs which contain a N-2-methoxyphenylpiperazine moiety are substrates for P-glycoprotein or other ABC transporters expressed at the blood-brain barrier, and that PET radiotracers containing this pharmacophore may display low brain uptake in rodents due to the action of these efflux transporters.
D3 receptors; PET; radiotracer; P-glycoprotein
Drug resistance is a significant problem in the treatment of ovarian cancer and can be caused by multiple mechanisms. Inhibition of apoptosis by the inhibitor of apoptosis proteins (IAPs) represents one such mechanism, and can be overcome by a mitochondrial protein called second mitochondria-derived activator of caspases (SMAC). We have previously shown that the ligands of sigma-2 receptors effectively induce tumor cell death. Additionally, because sigma-2 receptors are preferentially expressed in tumor cells, their ligands provide an effective mechanism for selective anti-cancer therapy.
In the current work, we have improved upon the previously described sigma-2 ligand SW43 by conjugating it to a pro-apoptotic small molecule SMAC mimetic SW IV-52, thus generating the novel cancer therapeutic SW IV-134. The new cancer drug was tested for receptor selectivity and tumor cell killing activity in vitro and in vivo.
We have shown that SW IV-134 retained adequate sigma-2 receptor binding affinity in the context of the conjugate and potently induced cell death in ovarian cancer cells. The cell death induced by SW IV-134 was significantly greater than that observed with either SW43 or SW IV-52 alone and in combination. Furthermore, the intraperitoneal administration of SW IV-134 significantly reduced tumor burden and improved overall survival in a mouse xenograft model of ovarian cancer without causing significant adverse effects to normal tissues. Mechanistically, SW IV-134 induced degradation of cIAP-1 and cIAP-2 leading to NF-қB activation and TNFα-dependent cell death.
Our findings suggest that coupling sigma-2 ligands to SMAC peptidomimetics enhances their effectiveness while maintaining the cancer selectivity. This encouraging proof-of-principle preclinical study supports further development of tumor-targeted small peptide mimetics via ligands to the sigma-2 receptor for future clinical applications.
Sigma-2 ligand; SMAC-peptidomimetic; Small molecule; Drug conjugate; Targeted drug delivery; Apoptosis; Ovarian cancer
A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group, and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both in vitro (i.e. recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells compared to the corresponding unsubstituted N-phenyl isatin analogs.
caspase-3; apoptosis; cell death
A series of fluorine containing N-(2-methoxyphenyl)piperazine and N-(2-fluoroethoxy)piperazine analogues were synthesized and their affinities for human dopamine D2, D3 and D4 receptors were determined. Radioligand binding studies identified five compounds, 18a, 20a, 20c, 20e and 21e, which bind with high affinity at D3 (Ki = 0.17 to 5 nM) and moderate to high selectivity for D3 vs. D2 receptors (ranging from ∼25 to 163-fold). These compounds were also evaluated for intrinsic activity at D2 and D3 receptors using a forskolin-dependent adenylyl cyclase assay. This panel of compounds exhibits varying receptor subtype binding selectivity and intrinsic activity at D2 vs. D3 receptors. These compounds may be useful for behavioral pharmacology studies on the role of D2-like dopamine receptors in neuropsychiatric and neurological disorders. Furthermore, compound 20e, which has the highest binding affinity and selectivity for the D3 receptor (Ki = 0.17 nM for D3, 163-fold selectivity for D3 vs. D2 receptors) represents a candidate fluorine-18 radiotracer for in vivo PET imaging studies on the regulation of D3 receptor expression.
IAP (inhibitor of apoptosis) proteins play a central role in many types of cancer, and IAP antagonists are in development as anti-cancer agents. IAP antagonists cause apoptosis in many cells, but they also activate alternative NF-κB signaling through NIK, which regulates osteoclasts. In bone metastasis, a positive feedback loop between tumors and osteoclasts promotes tumor growth and osteolysis. We therefore tested the effect of IAP antagonists on the bone microenvironment for metastasis. In both drug-sensitive and drug-resistant tumors, growth in bone was favored compared to other sites during IAP antagonist treatment. These drugs also caused osteoporosis and increased osteoclastogenesis, mediated by NIK, and enhanced tumor-associated osteolysis. Co-treatment with zoledronic acid, a potent osteoclast inhibitor, reduced IAP antagonist-enhanced tumor growth in bone and osteolysis. Thus, IAP-based cancer treatment may be compromised by osteoporosis and enhanced skeletal metastasis which may be prevented by anti-resorptive agents.
bone metastasis; IAP antagonist; osteoclast; NF-κB; NIK
15-(4-(2-[18F]fluoroethoxy)phenyl)pentadecanoic acid ([18F]7) was synthesized as a PET probe for assessing myocardial fatty acid metabolism. The radiosynthesis of [18F]7 was accomplished using a two step reaction, starting with the corresponding tosylate ester, methyl 15-(4-(2-(tosyloxy)ethoxy)phenyl)pentadecanoate (5) and gave the radiolabeled fatty acid, [18F]7 in a radiolabeling yield of 55 – 60% and a specific activity of > 2,000 Ci/mmol (decay corrected to EOB). The biological evaluation of [18F]7 in rats displayed high uptake in heart (1.94%.ID/g at 5 min), which was higher than the uptake (%ID/g) in blood, lung, muscle, pancreas and brain. MicroPET studies of [18F]7 in Sprague-Dawley rats demonstrated excellent images of the myocardium when compared with [11C]palmitate images in the same animal. Moreover, the tracer kinetics of [18F]7 paralleled those seen with [11C]palmitate, with an early peak followed by biphasic washout. When compared to [11C]palmitate, [18F]7 exhibited a slower early clearance (0.17 ± 0.01 vs. 0.30 ± 0.02, P < 0.0001) and a significantly higher late clearance (0.0030 ± 0.0005 vs. 0.0006 ± 0.00013, P < 0.01). These initial studies suggest that [18F]7 could be a potentially useful clinical PET tracer to assess abnormal myocardial fatty acid metabolism.
Fatty acid; PET imaging; F-18
Sigma-2 receptors represent an endogenous marker for proliferation in solid tumors. The high affinity, high selectivity σ2 receptor ligand N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-fluoroethoxy)-5-iodo-3-methoxybenzamide (3) was separately radiolabeled with F-18 and I-125. The radiolabeling yield was 30% and 70% for [18F]3 and [125I]3, respectively. Studies of [125I]3 using murine 66 breast tumor membrane homogenates and evaluation of [18F]3 and [125I]3 in 66 tumor-bearing mice indicate that this ligand has potential as a PET or a SPECT probe for imaging σ2 receptors in breast cancer.
Breast cancer; Molecular imaging; F-18; I-125; sigma receptor
We previously reported on the synthesis of substituted
indole analogues with nanomolar affinity at D2 dopamine receptors,
ranging from 10- to 100-fold selective for D2 compared to the D3 dopamine
receptor subtype. More recently, we evaluated a panel of aripiprazole
analogues, identifying several analogues that also exhibit D2 vs D3
dopamine receptor binding selectivity. These studies further characterize
the intrinsic efficacy of the compound with the greatest binding selectivity
from each chemical class, 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol
(SV 293) and 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one
(SV-III-130s), using an adenylyl cyclase inhibition assay, a G-protein-coupled
inward-rectifying potassium (GIRK) channel activation assay, and a
cell based phospho-MAPK (pERK1/2) assay. SV 293 was found to be a
neutral antagonist at D2 dopamine receptors using all three assays.
SV-III-130s is a partial agonist using an adenylyl cyclase inhibition
assay but an antagonist in the GIRK and phospho ERK1/2 assays. To
define the molecular basis for the binding selectivity, the affinity
of these two compounds was evaluated using (a) wild type human D2
and D3 receptors and (b) a panel of chimeric D2/D3 dopamine receptors.
Computer-assisted modeling techniques were used to dock these compounds
to the human D2 and D3 dopamine receptor subtypes. It is hoped that
these studies on D2 receptor selective ligands will be useful in the
future design of (a) receptor selective ligands used to define the
function of D2-like receptor subtypes, (b) novel pharmacotherapeutic
agents, and/or (c) in vitro and in vivo imaging agents.
Dopamine receptors; binding selectivity; functional
selectivity; GPCR structure; D2-like dopamine receptors; GPCR model building; ligand−receptor docking