Unlabeled Inhibitor Research Articles

Fluorescence polarization assay for screening FANCM-RMI inhibitors to target the alternative lengthening of telomeres.

Alternative Lengthening of Telomeres (ALT) is a mechanism used by 10-15% of all cancers to achieve replicative immortality, bypassing the DNA damage checkpoint associated with short telomeres that leads to cellular senescence or apoptosis. ALT does not occur in non-cancerous cells, presenting a potential therapeutic window for cancers where this mechanism is active. Disrupting the FANCM-RMI interaction has emerged as a promising therapeutic strategy that induces synthetic ALT lethality in genetic studies on cancer cell lines. There are currently no chemical inhibitors reported in the literature, in part due to the lack of reliable biophysical or biochemical assays to screen for FANCM-RMI disruption. Here we describe the development of a robust competitive fluorescence polarization (FP) assay that quantifies target binding at the FANCM-RMI interface. The assay employs a labeled peptide tracer TMR-RaMM2 derived from the native MM2 binding motif, which binds to recombinant RMI1-RMI2 and can be displaced by competitive inhibitors. We report the methods for recombinant production of RMI1-RMI2, design and evaluation of the tracer TMR-RaMM2, along with unlabeled peptide inhibitor controls to enable ALT-targeted drug discovery.

Structure-affinity and structure-residence time relationships of macrocyclic Gαq protein inhibitors

The macrocyclic depsipeptides YM-254890 (YM) and FR900359 (FR) are potent inhibitors of Gαq/11 proteins. They are important pharmacological tools and have potential as therapeutic drugs. The hydrogenated, tritium-labeled YM and FR derivatives display largely different residence times despite similar structures. In the present study we established a competition-association binding assay to determine the dissociation kinetics of unlabeled Gq protein inhibitors. Structure-affinity and structure-residence time relationships were analyzed. Small structural modifications had a large impact on residence time. YM and FR exhibited 4- to 10-fold higher residence times than their hydrogenated derivatives. While FR showed pseudo-irreversible binding, YM displayed much faster dissociation from its target. The isopropyl anchor present in FR and some derivatives was essential for slow dissociation. These data provide a basis for future drug design toward modulating residence times of macrocyclic Gq protein inhibitors, which has been recognized as a crucial determinant for therapeutic outcome.

Preclinical and first-in-human-brain-cancer applications of [18F]poly (ADP-ribose) polymerase inhibitor PET/MR.

BackgroundWe report preclinical and first-in-human-brain-cancer data using a targeted poly (ADP-ribose) polymerase 1 (PARP1) binding PET tracer, [18F]PARPi, as a diagnostic tool to differentiate between brain cancers and treatment-related changes.MethodsWe applied a glioma model in p53-deficient nestin/tv-a mice, which were injected with [18F]PARPi and then sacrificed 1 h post-injection for brain examination. We also prospectively enrolled patients with brain cancers to undergo dynamic [18F]PARPi acquisition on a dedicated positron emission tomography/magnetic resonance (PET/MR) scanner. Lesion diagnosis was established by pathology when available or by Response Assessment in Neuro-Oncology (RANO) or RANO-BM response criteria. Resected tissue also underwent PARPi-FL staining and PARP1 immunohistochemistry.ResultsIn a preclinical mouse model, we illustrated that [18F]PARPi crossed the blood–brain barrier and specifically bound to PARP1 overexpressed in cancer cell nuclei. In humans, we demonstrated high [18F]PARPi uptake on PET/MR in active brain cancers and low uptake in treatment-related changes independent of blood–brain barrier disruption. Immunohistochemistry results confirmed higher PARP1 expression in cancerous than in noncancerous tissue. Specificity was also corroborated by blocking fluorescent tracer uptake with an excess unlabeled PARP inhibitor in patient cancer biospecimen.ConclusionsAlthough larger studies are necessary to confirm and further explore this tracer, we describe the promising performance of [18F]PARPi as a diagnostic tool to evaluate patients with brain cancers and possible treatment-related changes.

Radioiodinated PARP1 tracers for glioblastoma imaging

BackgroundAlthough the understanding of the genetic and molecular basis of cancer has advanced significantly over the past several decades, imaging and treatment options for glioblastoma patients have been more limited (N Engl J Med 359:492-507, 2008). This is in part due to difficulties in diagnosing this disease early, combined with its diffuse, infiltrative growth. This study was aimed at the development of a novel diagnostic tool for glioblastoma through the synthesis of a small molecule based on radioiodinated poly(ADP-ribose)polymerase 1 (PARP1) targeted tracers. This PARP1 is a biomarker that is overexpressed in glioblastoma tissue, but has only low expression levels in the healthy brain (Neoplasia 16:432-40, 2014).MethodsA library of PARP1 inhibitors (iodo-PARPis) was synthesized. Based on their pharmacokinetic properties and nuclear PARP1 binding, the most successful inhibitor was radiolabeled with 131I and 124I. Biodistribution as well as imaging experiments were performed in orthotopic and subcutaneous mouse models of glioblastoma.ResultsOne member of our iodo-poly(ADP-ribose)polymerase 1 (PARP1) inhibitor library, I2-PARPi, shows promising biophysical properties for in vivo application. All synthesized tracers have IC50 values in the nanomolar range (9 ± 2–107 ± 4 nM) and were able to inhibit the uptake of a fluorescent PARP1 inhibitor analog (PARPi-FL). I2-PARPi was able to reduce the uptake of PARPi-FL by 78 ± 4 % in vivo. In mouse models of glioblastoma, we show that the radioiodinated inhibitor analog has high uptake in tumor tissue (U251 MG xenograft, tumor, 0.43 ± 0.06 %ID/g; brain, 0.01 ± 0.00 %ID/g; muscle, 0.03 ± 0.01 %ID/g; liver, 2.35 ± 0.57 %ID/g; thyroid, 0.24 ± 0.06 %ID/g). PET and SPECT imaging performed in orthotopic glioblastoma models with [124I]- and [131I]-I2-PARPi showed selective accumulation in the tumor tissue. These results were also verified using autoradiography of tumor sections, which displayed focal selective uptake of the tracer in the tumor regions as confirmed by histology. The uptake could be blocked through pre-injection of excess unlabeled PARP1 inhibitor (Olaparib).ConclusionsWe have successfully synthesized and radioiodinated the PARP1 selective tracer I2-PARPi. The novel tracer shows selective binding to tumor tissue, both in vitro and in models of glioblastoma, and has the potential to serve as a selective PET imaging agent for brain tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0123-1) contains supplementary material, which is available to authorized users.

Radiosynthesis and ex vivo evaluation of [(11)C-carbonyl]carbamate- and urea-based monoacylglycerol lipase inhibitors.

Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are the two primary enzymes that regulate the tone of endocannabinoid signaling. Although new PET radiotracers have been discovered for imaging FAAH in vivo, no such radiotracer exists for imaging MAGL. Here we report the radiosynthesis of five candidate MAGL radiotracers and their ex vivo evaluations in mice and rats. Candidate carbamate and urea MAGL inhibitors were radiolabeled at the carbonyl position by [(11)C]CO2 fixation. Radiotracers were administered (tail-vein injection) to rodents and brain uptake of radioactivity measured at early and late time points ex vivo. Specificity of uptake was explored by pretreatment with unlabeled inhibitors (2 mg/kg, ip) 30 min prior to radiotracer administration. All five candidate MAGL radiotracers were prepared in high specific activity (>65 GBq/μmol) and radiochemical purity (>98%). Moderate brain uptake (0.2-0.8 SUV) was observed for each candidate while pretreatment did not reduce uptake for four of the five tested. For two candidates ([(11)C]12 and [(11)C]14), high retention of radioactivity was observed in the blood (ca. 10 and 4 SUV at 40 min) which was blocked by pretreatment with unlabeled inhibitors. The most promising candidate, [(11)C]18, demonstrated moderate brain uptake (ca. 0.8 SUV) which showed circa 50% blockade by pretreatment with unlabeled 18. One putative and four reported potent and selective MAGL inhibitors have been radiolabeled via [(11)C]CO2 fixation as radiotracers for this enzyme. Despite the promising in vitro pharmacological profile, none of the five candidate radiotracers exhibited in vivo behavior suitable for PET neuroimaging.

Substrate- and Cell Contact-Dependent Inhibitor Affinity of Human Organic Cation Transporter 2: Studies with Two Classical Organic Cation Substrates and the Novel Substrate Cd2+

Polyspecific organic cation transporter Oct2 from rat (gene Slc22A2) has been previously shown to transport Cs(+). Here we report that human OCT2 (hOCT2) is able to transport Cd(2+) showing substrate saturation with a Michaelis-Menten constant (Km) of 54 ± 5.8 μM. Uptake of Cd(2+) by hOCT2 was inhibited by typical hOCT2 ligands (unlabeled substrates and inhibitors), and the rate of uptake was decreased by a point mutation in a substrate binding domain of hOCT2. Incubation of hOCT2 overexpressing human embryonic kidney 293 cells (HEK-hOCT2-C) or rat renal proximal tubule cells expressing rOct2 (NRK-52E-C) with Cd(2+) resulted in an increased level of apoptosis that was reduced by OCT2 inhibitory ligand cimetidine(+). HEK-hOCT2-C exhibited different functional properties when they were confluent or had been dissociated by removal of Ca(2+) and Mg(2+). Only confluent HEK-hOCT2-C transported Cd(2+), and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP(+)) by Cd(2+), MPP(+), tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd(2+) uptake, 4-[4-(dimethylamino)styryl]-N-methylpyridinium(+) (ASP(+)) uptake, and MPP(+) uptake using substrate concentrations far below the respective Km values. Employing a point mutation in the previously identified substrate binding site of rat Oct1 produced evidence that short distance allosteric effects between binding sites for substrates and inhibitors are involved in substrate-dependent inhibitor potency. Substrate-dependent inhibitor affinity is probably a common property of OCTs. To predict interactions between drugs that are transported by OCTs and inhibitory drugs, it is necessary to employ the specific transported drug rather than a model substrate for in vitro measurements.

FRI0389 RDEA3170, a novel, high affinity URAT1 inhibitor binds to a central domain within URAT1

BackgroundURAT1 inhibitors, used for the treatment of gout, lower serum urate levels by blocking uric acid reabsorption in the kidney proximal tubule through inhibition of the uric acid transporter URAT1....

Abstract 3910: Targeting urokinase plasminogen activator: evaluation of activity-based imaging probes in an orthotopic breast cancer model.

Abstract The urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. For the screening of uPA in breast tumors with commercially available ELISA kits that determine the total uPA content (active and inactive), fresh tumor tissue is required. Molecular imaging with activity-based uPA probes might overcome this limitation. Therefore we have developed some highly selective, non-peptidic and specific uPA activity-based probes based on the irreversible uPA inhibitor UAMC-00150. In this study we describe the evaluation of a fluorescent Cy5 labeled and a 18F labeled PET probe in the high uPA expressing MDA-MB-231 breast cancer mouse model to make the step towards clinical translation of this imaging biomarker. Orthotopic MDA-MB-231 tumors were established in nude mice and subjected to uPA imaging at a tumor volume of 150mm3. For the fluorescent imaging, three test groups were considered (n=5): mice from the first and the second experimental group received a single dose of equimolar amounts (0.23 μMol/kg) Cy5-uPA-probe or its inactivated hydrolyzed variant, respectively. The third group received an injection with unlabeled uPA inhibitor, 30 min before administration of Cy5-uPA probe, in a concentration that was 70-fold higher than the probe concentration. Mice were imaged at different time points for 48 h post injection (p.i.) and were subsequently sacrificed for ex vivo imaging and histology. PET imaging (n=15) was performed after i.v. injection of the radiotracer with a maximum of 0,2ml and/or 0,5mCi per injection, resulting in a range of 0,15-0,5mCi/injection. Animals were scanned at 15, 45, 90, 240 and 360 minutes p.i. At each time point, 3 mice were sacrificed to generate ex vivo biodistribution data. The Cy5-uPA-probe demonstrated good tumor-targeting properties in the high uPA-expressing breast tumor model, with fluorescent intensities reaching a maximum at 24 h post-probe administration in mice treated with Cy5-uPA-probe alone. The groups treated with the inactivated probe or the unlabeled inhibitor showed a significant decrease in the fluorescent tumor signal (p>0,016), which was also confirmed on histology. PET imaging with the 18F-uPA-probe showed a peak tumor uptake of 2.76 ± 0.37 %ID/g at 4 h p.i. Further in vitro PPB and ex vivo HPLC data revealed a high affinity of the tracer for blood proteins and a high metabolisation rate potentially explaining the rather moderate to low tumor uptake of the current PET uPa biomarker. In conclusion, fluorescent imaging data clearly indicate that the Cy5-uPA-probe enables non-invasive NIR-imaging of uPA expression in tumors in vivo. The first PET experiments indicate translational capabilities, for which we are now improving the pharmacokinetics of this and future uPa radiotracers. Citation Format: Johan Ides, Christel Vangestel, Jonas Messagie, Dieter Verzele, David Thomae, Sofie Thys, An Wouters, John-Paul Bogers, Sigrid Stroobants, Jurgen Joossens, Pieter Van der Veken, Marc Peeters, Filip Lardon, Steven Staelens, Koen Augustyns. Targeting urokinase plasminogen activator: evaluation of activity-based imaging probes in an orthotopic breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3910. doi:10.1158/1538-7445.AM2013-3910

Mechanistic Study of Dihydropteridine Reductase (DHPR) Using Raman Spectroscopy

York College of CUNY, Jamaica, NY, USA.Dihdyropteridine reductase (DHPR) catalyzes reduction of the unstable quinonoid dihydropteridine to the active tetrahydropteridine form through the oxidation of NADH. DHPR deficiency or any blockage in biosynthesis of tetrahydropteridine results in development of phenylketonuria (PKU), a progressive neurological illness which does not respond to dietary treatment. It has been speculated that DHPR or metabolites associated with it may have antioxidative properties. In another study, DHPR has been shown to have NADH-ferric reductase activity. This activity is postulated to have an important role in dietary iron uptake. There is an emerging role, though mechanistically unclear at this point, for BH4 in maintaining nitric oxide synthase (NOS) activity. NOS produces the signaling agent nitric oxide (•NO) from L-arginine. In the light of these emerging roles there is a growing need to completely understand how DHPR works.The mechanism of catalysis in DHPR is not fully understood. Previous studies have suggested the involvement of the thiol group of a cysteine residue in DHPR. DFT computational modeling of Raman spectra of the unlabeled and isolabeled inhibitor of DHPR provides valuable information for assigning Raman marker bands which are involved in enzyme- inhibitor interactions. Using Raman spectroscopic techniques, the effect of the inhibitors/substrate and/or cofactor on the protonation state of the thiol group is investigated. Raman spectroscopy is particularly effective because of a unique peak at ca. 2500 cm-1 due to the S-H stretch and another one at ca. 900 cm-1 due to the C-S bond. Peak shifts and modulation in intensities are monitored and analyzed in terms of its mechanistic implications.

Flow cytometric detection of prostate tumor cells using chemoaffinity labels

The enzyme-biomarker prostate-specific membrane antigen (PSMA) is an emerging target for imaging and therapeutic applications for prostate cancer. However, the use of PSMA for detecting circulating prostate tumor cells remains under-explored. The present study focuses on the specific labeling of PSMA+ prostate cancer cells with a fluorescent PSMA inhibitor and the quantitation of PSMA+ cells in blood by flow cytometry (FC) using a gating strategy to separate labeled PSMA+ cells from peripheral blood mononuclear cells. Suspensions of PSMA+ (LNCaP) and PSMA- (DU145) cells were incubated with the fluorescent PSMA inhibitor FAMX-CTT-54. Incubation parameters (time, temperature, and label concentration) were varied to optimize cell labeling. A gating protocol based on double fluorescent labeling of CD45 and PSMA was developed for the quantitiation of LNCaP cells in the presence of white blood cells from bovine blood. Nonfluorescent beads were added to the labeled cell mixture and served as internal standard for precise cellular quantification of LNCaP cells by flow cytometry. The fluorescent PSMA inhibitor FAMX-CTT-54 was specific for PSMA+ cells. The minimum time and concentration of FAMX-CTT-54 for effective labeling of PSMA+ cell suspensions at 37°C was 7.5 min and 35 nM, respectively; no labeling was observed on PSMA- cells. Co-incubation or pre-incubation of PSMA+ cells with the unlabeled PSMA inhibitor CTT-54 resulted in a concentration-dependent reduction in fluorescent labeling with FAMX-CTT-54 thereby confirming that the labeling was specific for PSMA. In blood samples in which LNCaP cells were added, an average of five cells were detected in a 115 µl sample of the most dilute sample examined (29 cells/ml); three cells were expected theoretically. The greater loss of labeling of PSMA+ cells with FAMX-CTT-54 when pre-incubated with CTT-54 is consistent with the irreversible mode of binding of CTT-54 to PSMA and subsequent internalization of the PSMA-inhibitor complex. The results suggest that fluorescent PSMA inhibitors can be utilized to effectively detect and quantify PSMA+ cells by FC. These results support the use of such compounds in the application of FC to detect, quantify, and characterize circulating prostate tumor cells.

The Human Cytomegalovirus UL36 Gene Controls Caspase-Dependent and -Independent Cell Death Programs Activated by Infection of Monocytes Differentiating to Macrophages

The cellular protease caspase-8 activates extrinsic apoptosis and also functions to promote monocyte-to-macrophage differentiation. Differentiation-induced alterations to antiviral caspase-8-dependent cell death pathways are unclear. Here, we show THP-1 monocyte-to-macrophage differentiation alters the specific cell death pathways activated in response to human cytomegalovirus (HCMV) infection. Employing viruses with mutations in UL36, the gene that encodes the viral inhibitor of caspase-8 activation (vICA), our data indicate that both caspase-dependent and -independent death pathways are activated in response to infection. Activation of caspase-dependent and -independent cell death responses restricted growth of vICA-deficient viruses, and vICA/pUL36 inhibited either response. Thus, these studies also reveal that the UL36 gene controls a caspase-independent cell death pathway. The impact of caspases on control of antiviral responses differed at early and late stages of macrophage differentiation. Early in differentiation, vICA-deficient virus-induced cell death was dependent on caspases and inhibited by the pan-caspase inhibitor z-VAD(OMe)-fluoromethyl ketone. In contrast, virus-induced death at late times of differentiation was caspase independent. Additional unlabeled and fluorescent inhibitors indicated that caspase-8 promoted death from within infected cells at early but not late stages of differentiation. These data highlight the multifunctional role of vICA/pUL36 as HCMV encounters various antiviral responses during macrophage differentiation.

A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: Important role for C-terminal biotin tagging of the kinase

Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR–FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S-transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases.

Internalization of cystatin C in human cell lines

Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A‐431, MCF‐7, MDA‐MB‐453, MDA‐MB‐468 and Capan‐1 cells internalized fluorophore‐conjugated cystatin C when exposed to physiological concentrations (1 μm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.

Single‐bead, Single‐molecule, Single‐cell Fluorescence

According to many current reports, the pharmaceutical business will hit a wall over the next few years. The generic competition is expected to wipe out a double-digit billion-dollar amount from top companies' annual sales between 2007 and 2012 (Wall Street Journal, online, December 6, 2007). The industry's science engine has stalled, new blockbusters are lacking, and patent expirations are a big problem. Also, the U.S. Food and Drug Administration is pulling back on approvals, requesting larger safety studies. Among the different approaches taken throughout the industry to improve productivity and to reduce the attrition rate of compounds in the drug discovery process, an extended application of quantitative biology and biophysical methods is ranked very high. Fluorescence spectroscopy and imaging represented the main detection technologies for assays and screening methods in recent years. Today, label-free detection methods, such as isothermal titration calorimetry, differential scanning calorimetry, tandem mass spectrometry (MS(n)), light scattering, or interferometry, start to provide viable alternative readouts for physicochemical characterization of leads and hit list triaging. However, the multidimensional nature of fluorescence along with its high sensitivity and single-molecule resolution remains an unparalleled source of molecular parameters to extract all different kinds of information on molecules and ligand-protein complexes in solution. Although fluorescence-based methods are currently applied throughout the different stages of the industrial drug discovery process, they are usually applied in an unconnected way. We have developed a fully integrated hit and lead discovery process combining bead-based synthesis and screening methods with confocal fluorescence microspectroscopy. The primary on-bead screening process provides fluorescent ligands that after a multistep characterization process ultimately leads to fully mechanistically characterized cellularly validated binders and inhibitors of target protein interactions. The unlabeled small-molecular inhibitors represent chemical starting points in drug discovery and target validation.

Properties of thiamin transport in isolated perfused hearts of chronically alcoholic guinea pigsThis article is one of a selection of papers published in the special issue Bridging the Gap: Where Progress in Cardiovascular and Neurophysiologic Research Meet.

The aim of this study was to determine the mechanism of transport of (14)C-thiamine in the hearts of healthy (nonalcoholic) and chronically alcoholic guinea pigs. We used the single-pass, paired-tracer dilution method on isolated and retrogradely perfused guinea pig hearts. The maximal cellular uptake (U(max)) and total cellular uptake (U(tot)) of (14)C-thiamine were determined under control conditions and under influence of possible modifiers. We tested how the presence of unlabeled thiamine, metabolic inhibitors, or absence of sodium ions influence the transport of (14)C-thiamine. The results of our experiments show that the transport of (14)C-thiamine is specific and energy-dependent and that its properties are significantly changed under the influence of chronic alcoholism. The latter effect occurs by increase in both U(max) and U(tot), as a manifestation of a compensatory mechanism in thiamine deficiency.

Lack of direct interaction between enalaprilat and the kinin B 1 receptors

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B 1 receptor (B 1R) contains a conserved HExxH motif also present in peptidases possessing a Zn 2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B 1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B 1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B 1R, enalaprilat, to address these questions using recombinant human B 1Rs and naturally expressed or recombinant ACE. [ 3H]Lys-des-Arg 9-BK bound to the human recombinant B 1Rs with high affinity ( K D 0.35 nM) in HEK 293a cells. [ 3H]Enalaprilat (0.25–10 nM) did not bind to cells expressing recombinant human B 1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B 1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B 1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B 1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B 1R stimulation in vivo.

Labeling of apoptotic JURL‐MK1 cells by fluorescent caspase‐3 inhibitor FAM‐DEVD‐fmk occurs mainly at site(s) different from caspase‐3 active site

Fluorochrome-labeled inhibitors of caspases (FLICA) have been designed as an alternative tool for the detection of caspase activation in whole cells. They should label the active site of the corresponding caspase through a covalent attachment to the reactive cysteine residue. One of the FLICAs, FAM-DEVD-fmk, was used to monitor apoptosis progression in leukemic JURL-MK1 cells by means of flow cytometry. The effects of unlabeled caspase inhibitors z-DEVD-fmk and z-VAD-fmk on FLICA staining were analyzed to evaluate the contribution of caspase-bound FLICA to the fluorescent signal. Covalent binding of inhibitors to caspase-3 subunit was revealed by Western blotting. Although the unlabeled inhibitors irreversibly bind to caspase-3, completely inhibit its activity, and prevent FLICA binding to caspase-3 even at concentrations lower than 5 muM, they have no effect on FLICA staining of apoptotic cells. Fluorescent signal of FLICA is characteristic for apoptotic cells but originates mainly from yet unspecified site(s) that differ from the caspase active site. This finding puts in doubt the specificity of staining by various FLICAs with regard to individual caspases and shows the need for an extreme care in the interpretation of data obtained using these labels.

Intra- or Intercomplex Binding to the γ-Secretase Enzyme: A MODEL TO DIFFERENTIATE INHIBITOR CLASSES

Intra- or Intercomplex Binding to the γ-Secretase Enzyme: A MODEL TO DIFFERENTIATE INHIBITOR CLASSES

A fluorescence polarization-based assay for peptidyl prolyl cis/ trans isomerase cyclophilin A

Peptidyl prolyl cis/ trans isomerase cyclophilin A (CypA) serves as a cellular receptor for the important immunosuppressant drug, cyclosporin A. In addition, CypA and its enzyme family have been found to play critical roles in a variety of biological processes, including protein trafficking, HIV and HCV infection/replication, and Ca 2+-mediated intracellular signaling. For these reasons, cyclophilins have emerged as potential drug targets for several diseases. Therefore, it is extremely important to screen for novel small molecule cyclophilin inhibitors. Unfortunately, the biochemical assays reported so far are not adaptable to a high-throughput screening format. Here, we report a fluorescence polarization-based assay for human CypA that can be adapted to high-throughput screening for drug discovery. The technique is based on competition and uses a fluorescein-labeled cyclosporin A analog and purified human CypA to quantitatively measure the binding capacity of unlabeled inhibitors. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with CypA is highly advantageous for the accurate assessment of inhibitor binding.

Binding of tritiated sildenafil, tadalafil, or vardenafil to the phosphodiesterase-5 catalytic site displays potency, specificity, heterogeneity, and cGMP stimulation.

Sildenafil, tadalafil, and vardenafil each competitively inhibit cGMP hydrolysis by phosphodiesterase-5 (PDE5), thereby fostering cGMP accumulation and relaxation of vascular smooth muscle. Biochemical potencies (affinities) of these compounds for PDE5 determined by IC(50), K(D) (isotherm), K(D) (dissociation rate), and K(D) ((1/2) EC(50)), respectively, were the following: sildenafil (3.7 +/- 1.4, 4.8 +/- 0.80, 3.7 +/- 0.29, and 11.7 +/- 0.70 nM), tadalafil (1.8 +/- 0.40, 2.4 +/- 0.60, 1.9 +/- 0.37, and 2.7 +/- 0.25 nM); and vardenafil (0.091 +/- 0.031, 0.38 +/- 0.07, 0.27 +/- 0.01, and 0.42 +/- 0.10 nM). Thus, absolute potency values were similar for each inhibitor, and relative potencies were vardenafil >> tadalafil > sildenafil. Binding of each (3)H inhibitor to PDE5 was specific as determined by effects of unlabeled compounds. (3)H Inhibitors did not bind to isolated PDE5 regulatory domain. Close correlation of EC(50) values using all three (3)H inhibitors competing against one another indicated that each occupies the same site on PDE5. Studies of sildenafil and vardenafil analogs demonstrated that higher potency of vardenafil is caused by differences in its double ring. Exchange-dissociation studies revealed two binding components for each inhibitor. Excess unlabeled inhibitor did not significantly affect (3)H inhibitor dissociation after infinite dilution, suggesting the absence of subunit-subunit cooperativity. cGMP addition increased binding affinity of [(3)H]tadalafil or [(3)H]vardenafil, an effect presumably mediated by cGMP binding to PDE5 allosteric sites, implying that either inhibitor potentiates its own binding to PDE5 in intact cells by elevating cGMP. Without inhibitor present, cGMP accumulation would stimulate cGMP degradation, but with inhibitor present, this negative feedback process would be blocked.