Functional assays to define agonists and antagonists of the sigma-2 receptor.

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC50 values of the sigma-2 ligands using the cell viability assay ranged from 11.4µM to >200µM, which were comparable with the EC50 values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.

Synthesis, radiolabeling and initial in vivo evaluation of [(11)C]KSM-01 for imaging PPAR-α receptors.

Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated nuclear receptor transcription factor that regulates the fatty acid ß-oxidation. An in vitro assay identified the p-methoxy phenyl ureido thiobutyric acid derivative KSM-01 (IC(50)=0.28±0.09nM) having a higher affinity to activate PPAR-α than the PPAR-α agonist GW7647 (IC(50)=0.46±0.19nM). In this study, we report the synthesis and initial in vivo evaluation of [(11)C]KSM-01. The radiosynthesis was carried out by first alkylating the corresponding p-phenol precursor with [(11)C]MeI in DMF using NaOH, followed by deprotection of the t-butyl ester group by TFA, yielding [(11)C]KSM-01. SUV analysis of dynamic micro PET/CT imaging data showed that [(11)C]KSM-01 accumulation was ∼2.0-fold greater in cardiac-specific PPAR-α overexpressing transgenic mice compared to wild-type littermates. The post-PET biodistribution studies were consistent with these results and demonstrated 2.5-fold greater radiotracer uptake in the heart of transgenic mice compared to the wild-type littermates. These results demonstrate the potential utility of PPAR-α agonists as PET radiopharmaceuticals.

Characterization and evaluation of two novel fluorescent sigma-2 receptor ligands as proliferation probes.

We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4',6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.

Synthesis and evaluation of isatin analogs as caspase-3 inhibitors: introduction of a hydrophilic group increases potency in a whole cell assay.

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.

[18F]- and [11C]-labeled N-benzyl-isatin sulfonamide analogues as PET tracers for apoptosis: synthesis, radiolabeling mechanism, and in vivo imaging study of apoptosis in Fas-treated mice using [11C]WC-98.

The radiolabeled isatin sulfonamide caspase-3 inhibitor, [18F] 2 (WC-II-89), is a potential PET radiotracer for noninvasive imaging of apoptosis. The radiolabeling mechanism was studied by 13C NMR, ESI/MS, and computational calculations. It was found that the high electrophilicity of the C3 carbonyl group in the isatin ring, which served as a trap for [18F]fluoride, was responsible for the failure of the radiolabeling via nucleophilic substitution of the mesylate group in 7a by [18F]fluoride. Once treated with a strong base, 7a opened the isatin ring completely to form an isatinate intermediate 16, which lost the ability to trap [18F]fluoride, thereby allowing the displacement of the mesylate group to afford the 18F-labeled isatinate 17. [18F] 17 can be converted to isatin [18F] 2 efficiently under acidic conditions. The ring-opening and re-closure of the isatin ring under basic and acidic conditions were confirmed by reversed phase HPLC analysis, ESI/MS and 13C NMR studies. Computational studies of model compounds also support the above proposed mechanism. Similarly, the ring-opening and re-closure method was used successfully in the synthesis of the 11C labeled isatin sulfonamide analogue [11C] 4 (WC-98). A microPET imaging study using [11C] 4 in the Fas liver apoptosis model demonstrated retained activity in the target organ (liver) of the treated mice. Increased caspase-3 activation in the liver was verified by the fluorometric caspase-3 enzyme assay. Therefore, this study provides a useful method for radio-synthesis of isatin derivative radiotracers for PET and SPECT studies, and [11C] 4 is a potential PET radiotracer for noninvasive imaging of apoptosis.

Isatin sulfonamide analogs containing a Michael addition acceptor: a new class of caspase 3/7 inhibitors.

A series of isatin sulfonamide analogs having a Michael acceptor were prepared and their potencies for inhibiting caspase-1, -3, -6, -7, and -8 were evaluated. These compounds have nanomolar potency for inhibiting the executioner caspases, caspase-3 and caspase-7, and have a low potency for inhibiting caspase-1, caspase-6, and caspase-8. The inhibition mechanism was investigated through NMR studies of the reaction between 11d and benzylmercaptan as a model for Cys-285 in the active site of caspase-3.

N-benzylisatin sulfonamide analogues as potent caspase-3 inhibitors: synthesis, in vitro activity, and molecular modeling studies.

A number of isatin sulfonamide analogues were prepared and their potencies for inhibiting caspase-1, -3, -6, -7, and -8 were evaluated in vitro. Several compounds displaying a nanomolar potency for inhibiting the executioner caspases, caspase-3 and caspase-7, were identified. These compounds were also observed to have a low potency for inhibiting the initiator caspases, caspase-1 and caspase-8, and caspase-6. Molecular modeling studies provided further insight into the interaction of this class of compounds with activated caspase-3. The results of the current study revealed a number of non-peptide-based caspase inhibitors that may be useful in assessing the role of inhibiting the executioner caspases in minimizing tissue damage in disease conditions characterized by unregulated apoptosis.

Synthesis and in vitro biological evaluation of pyrazole group-containing analogues for PDE10A.

Twenty eight new analogues were synthesized by optimizing the structure of MP-10 and their in vitro binding affinities towards PDE10A, PDE3A/B, and PDE4A/B were determined. Among these new analogues, 10a, 10b, 10d, 11a, 11b and 11d are very potent towards PDE10A and have IC50 values of 0.40 ± 0.02, 0.28 ± 0.06, 1.82 ± 0.25, 0.24 ± 0.05, 0.36 ± 0.03 and 1.78 ± 0.03 nM respectively; these six compounds displayed high selectivity for PDE10A versus PDE3A/3B/4A/4B. The promising compounds will be further validated in vivo to identify PDE10A imaging tracers.

Synthesis and evaluation of 18F-labeled PPARγ antagonists.

INTRODUCTION: 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. METHODS: 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. Micro-positron emission tomography (PET) imaging studies were performed in a transgenic mouse model having a heart-specific overexpression of PPARγ. RESULTS: 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 (SPA). However, only the chloro-based analogs (compounds 3 and 4) had activity in a whole-cell assay measuring activation of the PPARγ/retinoid X receptor complex. The microPET imaging studies in an MHC-PPARγ transgenic mouse model showed high uptake and PPARγ-specific binding for the irreversible antagonist [(18)F]3, whereas the corresponding reversible methoxy analog ([(18)F]5) displayed only nonspecific uptake in heart. CONCLUSIONS: The results of this preliminary study show that the irreversible antagonist [(18)F]3 may represent a novel strategy for imaging PPARγ in vivo with PET.

Radiolabeled isatin binding to caspase-3 activation induced by anti-Fas antibody.

INTRODUCTION: Noninvasive imaging methods that can distinguish apoptosis from necrosis may be useful in furthering our understanding of diseases characterized by apoptotic dysregulation as well as aiding drug development targeting apoptotic pathways. We evaluated the ability of radiolabeled isatins to quantify caspase-3 activity induced by the activation of the extrinsic apoptotic pathway by the anti-Fas antibody in mice. METHODS: The behavior of three different radiolabeled isatins ([(18)F]WC-II-89, [(18)F]WC-IV-3 and [(11)C]WC-98) was characterized in mice with and without anti-Fas antibody treatment by microPET imaging and biodistribution studies. The activity of [(18)F]WC-II-89 was also compared with [(99m)Tc]mebrofenin. The effect of pan-caspase inhibition with quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh) on [(18)F]WC-II-89 uptake was studied. Caspase-3 activity was confirmed by a fluorometric enzyme assay. RESULTS: All three tracers behaved similarly in microPET and biodistribution studies. Increased retention of all tracers was observed in the livers of treated animals and several other organs, all of which demonstrated increased caspase-3 enzyme activity; however, impaired hepatobiliary excretion made attribution of these findings to caspase-3 activity difficult. The isatin [(18)F]WC-II-89 was retained at statistically significantly higher levels in the organs after anti-Fas antibody treatment while [(99m)Tc]mebrofenin activity cleared, suggesting specific binding to activated caspase-3, but the magnitude of increased binding was still relatively low. Caspase inhibition with Q-VD-OPh partially blocked [(18)F]WC-II-89 retention but completely blocked caspase-3 enzyme activity in the liver. CONCLUSIONS: The radiolabeled isatins appear to bind specifically to caspase-3 in vivo, but their sensitivity is limited. Further optimization is required for these tracers to be useful for clinical applications.

Synthesis and in vitro evaluation of new analogues as inhibitors for phosphodiesterase 10A.

A series of analogues were synthesized by optimizing the structure of papaverine. The in vitro PDE10A binding affinity (IC(50)) values for these new analogues were measured; for compounds that have IC(50) value less than 60 nM for PDE10A, the binding affinities (IC(50) value) for PDE3A and PDE3B were tested. Of these analogues, compounds 6a, 6b, 6n, 8b, 8c and 11 displayed relatively higher PDE10A potency with IC(50) value in the range of 28-60 nM. The most potent compound 1-(4-(2-(2-fluoroethoxy)ethoxy)-3-methoxybenzyl)-6,7-dimethoxyisoquinoline (8c) has the IC(50) value of 28 ± 1.2 nM for PDE10A, 2200 ± 437 nM for PDE3A and 2520 ± 210 nM for PDE3B. Compared to papaverine, compound 8c displayed similar PDE10A potency but improved selectivity to PDE10A versus PDE3A and PDE3B. To identify high potent PDE10A inhibitor, further optimization of the structures of these analogues is necessary.

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 tumour 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 azide moiety and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was identified as PGRMC1 (progesterone receptor membrane component 1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalize 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.

Synthesis and in vitro evaluation of sulfonamide isatin Michael acceptors as small molecule inhibitors of caspase-6.

A key step in the onset of Huntington's disease is the caspase-6 mediated cleavage of the protein huntingtin into toxic fragments. Therefore, the inhibition of caspase-6 has been identified as a target for therapeutic drug development for the treatment of this disease. In this study, a series of isatin sulfonamide Michael acceptors having a high nanomolar potency for inhibiting caspase-6 and increased selectivity for caspase-6 versus caspase-3 inhibition is reported.

Design and synthesis of 2-amino-4-methylpyridine analogues as inhibitors for inducible nitric oxide synthase and in vivo evaluation of [18F]6-(2-fluoropropyl)-4-methyl-pyridin-2-amine as a potential PET tracer for inducible nitric oxide synthase.

A series of position-6 substituted 2-amino-4-methylpyridine analogues was synthesized and compounds 9, 18, and 20 were identified as the inhibitors with the greatest potential to serve as PET tracers for imaging inducible nitric oxide synthase (iNOS). [(18)F]9 was synthesized and evaluated in a mouse model of lipopolysaccharide (LPS)-induced iNOS activation. In vivo biodistribution studies of [(18)F]9 indicate higher tracer uptake in the lungs of the LPS-treated mice when compared to control mice. Tracer uptake at 60 min postinjection was reduced in a blocking study using a known inhibitor of iNOS. The expression of iNOS was confirmed by Western blot analysis of lung samples from the LPS-treated mice. MicroPET studies also demonstrated accumulation of radiotracer in the lungs of the LPS-treated mice. Taken collectively, these data suggest that [(18)F]9 shows favorable properties as a PET tracer to image iNOS activation with PET.

Comparison of radiolabeled isatin analogs for imaging apoptosis with positron emission tomography.

INTRODUCTION: Caspase-3 is one of the executioner caspases activated as a result of apoptosis. Radiolabeled isatins bind to caspase-3 with high affinity and are potential tracers for use with positron emission tomography to image apoptosis. We compared the ability of two novel radiolabeled isatins, [18F]WC-IV-3 and [11C]WC-98, to detect caspase-3 activation in a rat model of cycloheximide-induced liver injury. METHODS: Male Sprague-Dawley rats were treated with cycloheximide and then imaged with microPET 3 h later with [18F]WC-IV-3 and [11C]WC-98. Biodistribution studies were also performed simultaneously, with caspase-3 activation verified by fluorometric enzyme assay and Western blots. RESULTS: MicroPET imaging studies demonstrated similar behavior of both tracers but with a lower maximum peak with [11C]WC-98 than with [18F]WC-IV-3. Biodistribution studies demonstrated increased uptake of both tracers in the liver and spleen, but this was statistically significant only in the liver with both compounds. The level of [18F]WC-IV-3 uptake appeared to correlate roughly with rates of caspase-3 activation by the enzyme assay, but the magnitude of difference between treated and control groups was lower than that observed in previously published data with [18F]WC-II-89, another radiolabeled isatin analog. Activation was also confirmed in the liver and spleen but not in fat by Western blot. CONCLUSION: [18F]WC-IV-3 uptake appears to correlate with increased caspase-3 enzyme activity, but the dynamic range of uptake of these two tracers appears to be less than that seen with [18F]WC-II-89. Studies are ongoing to verify these results in other animal models of apoptosis.

Synthesis, radiolabeling, and in vivo evaluation of an 18F-labeled isatin analog for imaging caspase-3 activation in apoptosis.

A non-peptide-based isatin sulfonamide analog, WC-II-89, was synthesized and its inhibition toward recombinant human caspase-3 and other caspases was determined. This compound showed high potency for inhibiting caspase-3 and -7, and high selectivity against caspases-1, -6, and -8. [(18)F]WC-II-89 was synthesized via a nucleophilic substitution of the corresponding mesylate precursor in high yield and radiochemical purity. Biodistribution studies using [(18)F]WC-II-89 revealed higher uptake in liver and spleen of cycloheximide-treated rats, an animal model of apoptosis, relative to control animals. Western blot analysis confirmed the presence of activated caspase-3 in the liver and spleen of cycloheximide-treated animals. MicroPET imaging studies revealed a high uptake of the radiotracer in the liver of a cycloheximide-treated rat relative to the untreated control. These data suggest that [(18)F]WC-II-89 is a potential radiotracer for imaging caspase-3 activation in tissues undergoing apoptosis.