Scintillation Proximity Assay Research Articles

APOE alters glucose flux through central carbon pathways in astrocytes

The Apolipoprotein E (APOE) gene is a major genetic risk factor associated with Alzheimer's disease (AD). APOE encodes for three main isoforms in humans (E2, E3, and E4). Homozygous E4 individuals have more than a 10-fold higher risk for developing late-onset AD, while E2 carriers are protected. A hallmark of AD is a reduction in cerebral glucose metabolism, alluding to a strong metabolic component in disease onset and progression. Interestingly, E4 individuals display a similar regional pattern of cerebral glucose hypometabolism decades prior to disease onset. Mapping this metabolic landscape may help elucidate the underlying biological mechanism of APOE-associated risk for AD. Efficient metabolic coupling of neurons and glia is necessary for proper neuronal function, and disruption in glial energy distribution has been proposed to contribute to neuronal cell death and AD pathology. One important function of astrocytes – canonically the primary source of apolipoprotein E in the brain – is to provide metabolic substrates (lactate, lipids, amino acids and neurotransmitters) to neurons. Here we investigate the effects of APOE on astrocyte glucose metabolism in vitro utilizing scintillation proximity assays, stable isotope tracer metabolomics, and gene expression analyses. Glucose uptake is impaired in E4 astrocytes relative to E2 or E3 with specific alterations in central carbon metabolism. Using stable isotope labeled glucose [U-13C] allowed analyses of astrocyte-specific deep metabolic networks affected by APOE, and provided insight to the effects downstream of glucose uptake. Enrichment of 13C in early steps of glycolysis was lowest in E4 astrocytes (highest in E2), while synthesis of lactate from glucose was highest in E4 astrocytes (lowest in E2). We observed an increase in glucose flux through the pentose phosphate pathway (PPP), with downstream increases in gluconeogenesis, lipid, and de novo nucleotide biosynthesis in E4 astrocytes. There was also a marked increase in 13C enrichment in the TCA cycle of E4 astrocytes – whose substrates were also incorporated into biosynthetic pathways at a higher rate. Pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) are the two main enzymes controlling pyruvate entry to the TCA cycle. PC gene expression is increased in E4 astrocytes and the activity relative to PDH was also increased, compared to E2 or E3. Decreased enrichment in the TCA cycle of E2 and E3 astrocytes is suggestive of increased oxidation and non-glucose derived anaplerosis, which could be fueling mitochondrial ATP production. Conversely, E4 astrocytes appear to increase carbon flux into the TCA cycle to fuel cataplerosis. Together, these data demonstrate clear APOE isoform-specific effects on glucose utilization in astrocytes, including E4-associated increases in lactate synthesis, PPP flux, and de novo biosynthesis pathways.

Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay.

Over the last decades, the use of heavy-chain-only antibodies has received growing attention in academia and industry as research and diagnostic tools as well as therapeutics. Their generation has improved with the help of innovative new methods such as the sybody technology; however, identifying conformation-selective compounds against membrane proteins remains a major challenge. In this chapter, we apply a thermal shift scintillation proximity assay (SPA-TS) to identify sybodies from an in vitro display campaign with the ability to selectively stabilize the inhibitor-bound conformation of the human solute carrier (SLC) family transporter SC6A9 (GlyT1). Using detergent-purified GlyT1 protein and a tritium-labeled glycine uptake inhibitor small molecule, we find sybody candidates that increase the apparent melting temperature in SPA-TS by several degrees. The thermal shift stabilizes the GlyT1-inhibitor complex and qualifies the sybodies for structural studies and inhibitor-selective small molecule screening assays. The SPA-TS assay in its current form is adaptable to any antibody discovery campaign for membrane proteins and permits the generation of highly valuable tools in most stages of drug discovery and development.

Glutaminyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization, structure, and development as a screening platform.

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcatobs , governing interactions with tRNA, were 1.0 μM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.

Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: Selective biased agonism via Gαi1-proteins

Serotonin 5-HT2A receptors (5-HT2ARs) have been implicated in schizophrenia. However, postmortem studies on 5-HT2ARs expression and functionality in schizophrenia are scarce. The 5-HT2AR mRNA and immunoreactive protein expression were evaluated in postmortem tissue from dorsolateral prefrontal cortex (DLPFC) of antipsychotic-free (n = 18) and antipsychotic-treated (n = 9) subjects with schizophrenia, and matched controls (n = 27). Functional coupling of 5-HT2AR to G-proteins was tested by measuring the activation induced by the agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride ((±)DOI) in antibody-capture [35S]GTPγS scintillation proximity assays (SPA). In antipsychotic-free schizophrenia subjects, 5-HT2AR mRNA expression and protein immunoreactivity in total homogenates was similar to controls. In contrast, in antipsychotic-treated schizophrenia subjects, lower mRNA expression (60±9% vs controls) and a trend to reduced protein immunoreactivity (86±5% vs antipsychotic-free subjects) just in membrane-enriched fractions was observed. [35S]GTPγS SPA revealed a significant ~6% higher stimulation of Gαi1-protein by (±)DOI in schizophrenia, whereas activation of the canonical Gαq/11-protein pathway by (±)DOI remained unchanged. Expression of Gαi1- and Gαq/11-proteins did not differ between groups. Accordingly, in rats chronically treated with clozapine, but not with haloperidol, a 30–40% reduction was observed in 5-HT2AR mRNA expression, 5-HT2AR protein immunoreactivity and [3H]ketanserin binding in brain cortical membranes. Overall, the data suggest a supersensitive 5-HT2AR signaling through inhibitory Gαi1-proteins in schizophrenia. Together with previous results, a dysfunctional pro-hallucinogenic agonist-sensitive 5-HT2AR conformation in postmortem DLPFC of subjects with schizophrenia is proposed. Atypical antipsychotic treatment would contribute to counterbalance this 5-HT2AR supersensitivity by reducing receptor expression.

Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene (lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s-1, resulting in kcatobs/KM values of 0.29, 0.036, and 0.11 s-1µM-1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.

Cannabinoid receptor antagonists and G protein subtypes

The therapeutic importance of inverse agonism for cannabinoid receptors (CB1R) has been proposed. The underlying mechanisms of inverse cannabimimetic effects by the CB1R antagonist/inverse agonist, SR141716 were investigated in both N18TG2 mouse neuroblastoma and human embryonic kidney (HEK)‐293 cells stably expressing CB1Rs (CB1‐HEK). We utilized cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) (Eldeeb et al., 2016 and 2017). The CB1R agonist, CP55940 stimulated [35S]GTPγS binding (from 1nM: 2.6±5.5% to 1μM: 54±7.3% over basal). Co‐incubation with 1μM of SR141716 inhibited CP55940‐mediated [35S]GTPγS binding to −27±3.2% and −5.6±7%, respectively. In N18TG2 cells treated with the diacylglycerol lipase inhibitor, tetrahydrolipstatin (THL, orlistat), 1μM for 2h, SR141716 antagonizing effects were unchanged. In membranes from cells pretreated with pertussis toxin (PT) (100 ng/ml) for 20 h, SR14171 inhibition of [35S]GTPγS binding was attenuated in PT‐pretreated membranes, indicating that SR141716 effects were Gi/o protein‐dependent. Using SPA, SR14176 alone inhibited [35S]GTPγS binding by Gi/o protein subtypes, primarily Gαi1 and Gαi3 (−46.5±6.6% and −46.7±6.2% with respect to basal). Both previously reported CB1R neutral antagonists, AM4113 and PIMSR, at 1μM were able to reduce maximal CP55940‐stimulated [35S]GTPγS binding for total and individual Gi/o protein subtypes, primarily Gαi1 and Gαi3. Neither AM4113 nor PIMSR (1μM) were able to competitively reverse the SR141716‐mediated inhibition of [35S]GTPγS binding in N18TG2 neuronal cells membranes. In CB1‐HEK cell membranes neither AM4113 nor PIMSR(1μM) behaved as neutral antagonist to the CP55940‐mediated stimulation of [35S]GTPγS binding. The CB1R constitutive activity is mediated by Gi/o protein stimulation, predominantly Gαi1 and Gαi3, and in our experiments, was not associated with endogenously released endocannabinoids. Further research into the molecular mechanism of the competitive antagonist inverse agonist and/or negative allosteric modulator effects of SR141716 and its analogs will be necessary to consider in developing “neutral antagonists” for the CB1 receptor.Support or Funding InformationNIH grants R01‐DA03690, R01‐DA042157, P50‐DA006634, K12‐GM102773This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Inhibitors of Glutaminyl‐tRNA synthetase from Pseudomonas aeruginosa

IntroductionAntibiotic resistance is a growing global threat and infections caused by resistant bacteria have become increasingly difficult to treat with most current antibiotics. Pseudomonas aeruginosa is a Gram‐negative opportunistic pathogen and is the leading cause of mortality in cystic fibrosis patients. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. The glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was over‐expressed, enzymatically characterized and developed into a screening platform for the discovery of chemical compounds that inhibit protein synthesis.MethodsThe P. aeruginosa GlnRS was developed, using scintillation proximity assay (SPA) technology, as a platform to screen chemical compounds for detection of compounds that inhibited function. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with inhibitory activity.ResultsFrom this screening campaign three compounds (BM02E04, BM04B05, and BM04H03) were identified and confirmed as inhibitors of the activity of P. aeruginosa GlnRS. BM02E04, BM04B05, and BM04H03 were observed to inhibit the activity of GlnRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. The minimum inhibitory concentration (MICs) was determined against a panel of bacteria including; E. coli, E. coli tolC mutant, E. faecalis, H. influenzae, M. cattarrhalis, P. aeruginosa, P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain, S. aureus, and S. pneumoniae. There was no activity observed against the wild‐type strain of E. coli or P. aeruginosa, but broad spectrum activity was observed against other Gram‐negative and Gram‐positive bacteria. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus and M. catarrhalis. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, there was no toxicity observed by either BM02E04 or BM04B05 at any concentration tested up to 400 μg/ml.ConclusionP. aeruginosa GlnRS was cloned, over‐expressed, characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms. Three compounds were identified as having inhibitory activity against the aminoacylation activity of GlnRS. Two of the compounds were further characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Characterization Chemical Compounds that Inhibit Lysyl‐tRNA Synthetase from Pseudomonas aeruginosa

IntroductionPseudomonas aeruginosa is an opportunistic pathogen and a major cause of nosocomial infections. The enhanced ability of P. aeruginosa to acquire resistance to most antibiotics places it in a special class of pathogens, the so‐called “superbugs”. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis and are validated targets for development of antibacterial agents. The gene encoding P. aeruginosa lysyl‐tRNA synthetase (LysRS) was cloned and the resulting overexpressed protein was purified to 98% homogeneity.Methods and ResultsAminoacylation assays were used to measure kinetic parameters for interactions of LysRS with tRNA and ATP:PPi assays were used to determine the kinetic parameters for interactions with lysine and ATP. The KM values for interaction with lysine, ATP and tRNALys were determined to be 45.3, 627, and 3.3 μM, respectively. The kcatobs values were calculated to be 13.3, 22.8, and 0.35 sec−1. This resulted in kcatobs/KM values of 0.29, 0.036, 0.11 s−1μM−1. P. aeruginosa LysRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen natural product (800) and synthetic (890) compound libraries to identify compounds with the ability to inhibit the function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified and inhibitory activity was confirmed. These compounds inhibited the ability of LysRS to aminoacylate tRNALys with IC50 values of 35.3, 66.9, and 36.4 μM, respectively. The minimum inhibitory concentration (MIC) was determined for each compound against a panel of ten pathogenic bacteria. Each compound exhibited broad spectrum activity. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus, however in cultures of M. catarrhalis, BT06F11 and BT08F04 was observed to be bactericidal. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, BT06F11 was not toxic at any concentration tested. BM01D09 was observed to only inhibit human cell growth at high concentrations (CC50 = 370 μM). However, BT08F04 was toxic at much lower concentrations (CC50 = 61 μM).ConclusionLysRS from P. aeruginosa was characterized and developed into a screening platform to identify potential anti‐bacterial compounds. Three compounds were identified as having inhibitory activity against the aminoacylation of LysRS. The compounds were then characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Core‐Shell Nanoparticle Probe Scintillation Proximity Assays for Biological Samples

To achieve the overall goal of sensitive and selective radioisotope detection in aqueous samples, we have developed a novel polystyrene‐core silica‐shell nanoparticle scintillator platform (nanoSPA). nanoSPA enabled separation‐free quantification of radiolabeled analytes in complex samples as demonstrated by scintillation proximity assays via protein‐tag, biotin, and antibody binding as well as by other methods such as chemical crosslinking and adsorption.β‐particle emitting radionuclides such as 3H, 33P, and 35S are useful molecular labels due to their small size but are challenging to detect and quantify with spatial and temporal resolution in biological samples due to their low energies (Emax ≤ 300 keV) and short penetration depths (≤ 0.6 mm). Activity measurements for these β‐emitters are usually made in milliliter volumes of liquid scintillation cocktail (LSC), a mixture of energy‐absorbing organic solvents, surfactants, and scintillant fluorophores, which is incompatible with living cells and dynamic biological measurements. Alternatively, solid polymer or inorganic crystals can be used, but the orientation of polymer sheets, the large size of polymer particles (> 2μm) and the high density of inorganic scintillators can result in inaccurate counting. nanoSPA avoids the toxicity of LSC as well as many of the limitations of polymer and inorganic crystal scintillators. The polystyrene acts as an absorber for energy from emitted β‐particles and is loaded with scintillant fluorophores to which the energy is transferred, leading to photon emission at visible wavelengths. The silica shell serves as a hydrophilic shield for the polystyrene core and facilitates functionalization with specific binding molecules for scintillation proximity assays. In this way, the short penetration depths of low‐energy β‐particles are used advantageously, as radiolabeled analytes that are not close enough to the nanoparticle surface (primarily bound to the surface during an assay) are less likely to be detected, resulting in low background signal. Selectivity was determined to be up to 30 times greater for bound 3H‐analytes over unbound 3H analytes when the labeled species was covalently bound to nanoSPA, 18 times greater for antibody functionalized nanoSPA, 8 times greater for biotin functionalized nanoSPA, and 4 times greater for ssDNA functionalized nanoSPA. We have also measured and monitored peptide phosphorylation with kinase and 33P ɣ ‐ ATP. nanoSPA not only facilitates measurement of radiolabeled analytes in bulk aqueous solutions, but due to the small diameter and the protection of the hydrophobic polymer core by the silica shell, nanoSPA particles could potentially be used as cellular or intracellular imaging probes.Support or Funding InformationThis work was supported in part by the National Science Foundation under grant number 1807343, the National Institutes of Health via the National Institute of Biomedical Imaging and Bioengineering under grant number R21EB019133 and the National Institute of General Medical Sciences under grant number 1R01GM116946.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cloning and Characterization of the Tyrosyl‐S and Tyrosyl‐Z tRNA Synthetases from Pseudomonas aeruginosa

IntroductionPseudomonas aeruginosa, a Gram‐negative opportunistic pathogen, is a leading cause of nosocomial infections. The ability of this bacteria to form resilient biofilms on implanted medical devices results in high mortality rates. Patients with cystic fibrosis (CF) are especially at risk as P. aeruginosa colonizes the lungs of these patients forming biofilms which leads to chronic infections and is a leading cause of death. Aminoacyl‐tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to their cognate tRNAs. These enzymes are essential for protein synthesis in bacteria and serve as validated targets for the development of new anti‐infectives. Unlike in other bacteria, the P. aeruginosa genome contains two genes (tyrS and tyrZ) which encode two distinct TyrRS enzymes, which are very different at the amino acid level containing only 41.1/26.8 percent similar/conserved residues.Methods and ResultsThe gene encoding P. aeruginosa TyrRS‐Z was cloned and overexpressed in E. coli cells, and purified to homogeneity. Amplification of tyrS by PCR was unsuccessful, therefore the gene sequence was optimized for codon expression and synthetically synthesized. Cloning of the optimized gene resulted in expression of large amounts of TyrRS‐S. Both forms of TyrRS were active in aminoacylation. The kinetic parameters for the interaction of these enzymes with their three substrates (tRNA, ATP, tyrosine) were determined using timed aminoacylation and ATP:PPi exchange assays. Both forms of TyrRS were developed into screening platforms using scintillation proximity assay (SPA) technology. Using this assay, a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds was screened to detect compounds with the ability to inhibit the function of TyrRS. A number of inhibitory compounds were confirmed and are currently being characterized for the ability to inhibit both the enzymatic activity (IC50) of both forms of TyrRS and growth (MIC) of a panel of problematic pathogens in culture. They are also being analyzed to determine the potential mechanism of inhibition relative to substrate binding, global mode of action of inhibition of growing cultures of bacteria, as well as toxicity to human cell cultures.ConclusionTyrRS‐S and TyrRS‐Z from P. aeruginosa were cloned, expressed, characterized and used for development of screening platforms to identify compounds that have the potential for development as antibacterial agents against drug resistant pathogenic organisms.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hybrid Nanoparticle Platform for Nanoscale Scintillation Proximity Assay.

β-particle emitting radionuclides, such as 3H, 14C, 32P, 33P, and 35S, are important molecular labels due to their small size and the prevalence of these atoms in biomolecules but are challenging to selectively detect and quantify within aqueous biological samples and systems. Here, we present a core-shell nanoparticle-based scintillation proximity assay platform (nanoSPA) for the separation-free, selective detection of radiolabeled analytes. nanoSPA is prepared by incorporating scintillant fluorophores into polystyrene core particles and encapsulating the scintillant-doped cores within functionalized silica shells. The functionalized surface enables covalent attachment of specific binding moieties such as small molecules, proteins, or DNA that can be used for analyte-specific detection. nanoSPA was demonstrated for detection of 3H-labeled analytes, the most difficult biologically relevant β-emitter to measure due to the low energy β-particle emission, using three model assays that represent covalent and non-covalent binding systems that necessitate selectivity over competing 3H-labeled species. In each model, nmol quantities of target were detected directly in aqueous solution without separation from unbound 3H-labeled analyte. The nanoSPA platform facilitated measurement of 3H-labeled analytes directly in bulk aqueous samples without surfactants or other agents used to aid particle dispersal. Selectivity for bound 3H-analytes over unbound 3H analytes was enhanced up to 30-fold when the labeled species was covalently bound to nanoSPA, and 4- and 8-fold for two non-covalent binding assays using nanoSPA. The small size and enhanced selectivity of nanoSPA should enable new applications compared to the commonly used microSPA platform, including the potential for separation-free, analyte-specific cellular or intracellular detection.

Ultrasound-enhanced scintillation proximity assay for rapid diagnostics

Scintillation proximity assay (SPA) is a type of radioimmunoassay (RIA). We apply ultrasound enhancement to the general SPA. All assay procedures, including the antibody coating and radiolabeled antigen binding are achieved by simply mixing then standing for 5 min in an ultrasound chamber. No additional incubation time is required. To further demonstrate the capability of the UE-SPA, a quantitative measurement of CD55 in various grades of colon tumors was assessed on human tissue slides. The results showed a significant correlation between CD55 expression and tumorigenesis. In conclusion, we confirmed that UE-SPA is a reliable, rapid and alternative to RIA.

Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform.

Pseudomonas aeruginosa is an opportunistic multi-drug resistant pathogen implicated as a causative agent in nosocomial and community acquired bacterial infections. The gene encoding prolyl-tRNA synthetase (ProRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. ProRS was kinetically evaluated and the KM values for interactions with ATP, proline, and tRNA were 154, 122, and 5.5 μM, respectively. The turn-over numbers, kcatobs , for interactions with these substrates were calculated to be 5.5, 6.3, and 0.2 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa ProRS was solved to 2.60 Å resolution. The amino acid sequence and X-ray crystal structure of P. aeruginosa ProRS was analyzed and compared with homologs in which the crystal structures have been solved. The amino acids that interact with ATP and proline are well conserved in the active site region and overlay of the crystal structure with ProRS homologs conforms to a similar overall three-dimensional structure. ProRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 890 chemical compounds, resulting in the identification of two inhibitory compounds, BT06A02 and BT07H05. This work confirms the utility of a screening system based on the functionality of ProRS from P. aeruginosa.

Higher lipid turnover and oxidation in cultured human myotubes from athletic versus sedentary young male subjects

In this study we compared fatty acid (FA) metabolism in myotubes established from athletic and sedentary young subjects. Six healthy sedentary (maximal oxygen uptake (VO2max) ≤ 46 ml/kg/min) and six healthy athletic (VO2max > 60 ml/kg/min) young men were included. Myoblasts were cultured and differentiated to myotubes from satellite cells isolated from biopsy of musculus vastus lateralis. FA metabolism was studied in myotubes using [14C]oleic acid. Lipid distribution was assessed by thin layer chromatography, and FA accumulation, lipolysis and re-esterification were measured by scintillation proximity assay. Gene and protein expressions were studied. Myotubes from athletic subjects showed lower FA accumulation, lower incorporation of FA into total lipids, triacylglycerol (TAG), diacylglycerol and cholesteryl ester, higher TAG-related lipolysis and re-esterification, and higher complete oxidation and incomplete β-oxidation of FA compared to myotubes from sedentary subjects. mRNA expression of the mitochondrial electron transport chain complex III gene UQCRB was higher in cells from athletic compared to sedentary. Myotubes established from athletic subjects have higher lipid turnover and oxidation compared to myotubes from sedentary subjects. Our findings suggest that cultured myotubes retain some of the phenotypic traits of their donors.

Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation.

The human dopamine transporter (hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the structural studies of hDAT have proven difficult. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, we identified ligands that stabilize hDAT in detergent micelles. We then screened ~200 single residue mutants of hDAT using a high-throughput scintillation proximity assay and identified a thermostable variant (I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.

Abstract LB-068: Development of a screening cascade to identify selective small molecule inhibitors of DNMT1

Abstract DNA Methyltransferases (or DNMTs) play an important role in genomic integrity. The DNMT family of enzymes catalyse the transfer of methyl groups to specific CpG structures on DNA, with DNMT1 acting as a maintenance methyltransferase and DNMT3a and 3b acting as de novo methyltransferases. Upon cell division, epigenetic profiles within DNA strands are copied, with DNMT1 ensuring that methyl marks are transferred correctly to the newly synthesised DNA strand, thereby conferring heritability. Aberrant DNA hypermethylation within DNA promoter regions can lead to gene silencing, which is a hallmark of human cancer. Treatment with hypomethylating agents such as azacytidine and decitabine reverse methyl marks and show clinical benefit for the treatment of Acute Myeloid Leukemia (AML) and Chronic Myelomonocytic Leukemia (CMML) and are also the standard of care for Myelodysplastic Syndrome (MDS). However, these agents also show dose limiting toxicity due to their irreversible and non-selective mechanism of action and generally poor pharmacokinetic properties. Over the past decades both the Pharma and Biotech industries have deployed significant resource in a bid to identify and develop potent, selective DNMT1 inhibitors. These attempts have delivered little, if any, success until now. Collaboration between the CRUK Manchester Institute and GSK has resulted in the successful development of a screening cascade to identify compounds that selectively inhibit DNMT1. A collection of over 1 million compounds was screened at GSK using a radioactive scintillation proximity assay (SPA). Preliminary hits were identified and triaged through our screening cascade to determine genuine hits from false positive results. Firstly, ‘hits' from the SPA screen were tested in rapid-fire mass spectrometry and also in fluorescence intensity coupled ‘break-light' assays. Though these orthogonal screens apparently confirmed activity, IC50 screening of many of these compounds revealed high Hill slopes and high maximal inhibition profiles which are indicative of non-specific inhibition mechanisms. Detailed mechanism of inhibition studies alongside differential scanning fluorimetry was employed to rule out those compounds that were binding non-specifically to the oligonucleotide substrate. One series of compounds displayed IC50 profiles consistent with specific inhibition of DNMT1, with a 1:1 stoichiometry and no evidence for non-specific binding to the oligonucleotide substrate. Selectivity screening against DNMT3a and 3b also showed that within the DNMT family this series of compounds was selective for DNMT1 only. All compounds that displayed reasonable biochemical potency, (sub 1µΜ), were tested further in cellular assays to confirm target engagement. Through our extensive range of assays and thorough profiling of hit compounds, we were able to quickly de-validate the significant number of false positive hits that have plagued previous drug discovery screens in this area and identify a series of genuine, selective, small molecule inhibitors of DNMT1. Citation Format: Alexandra Stowell, Graeme Thomson, Mark Cockerill, Charlotte Burt, Emma Fairweather, Ian Waddell, Ali Raoof, Allan Jordan, Donald 0gilvie, Melissa Pappalardi, Juan Luengo, Mehul Patel, Ryan Kruger, Chris Carpenter. Development of a screening cascade to identify selective small molecule inhibitors of DNMT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-068.

Identification of a novel selective small-molecule inhibitor of protein arginine methyltransferase 5 (PRMT5) by virtual screening, resynthesis and biological evaluations

As one of the most promising anticancer target in protein arginine methyltransferase (PRMT) family, PRMT5 has been drawing more and more attentions, and many efforts have been devoted to develop its inhibitors. In this study, three PRMT5 inhibitors (9, 16, and 23) with novel scaffolds were identified by performing pharmacophore- and docking-based virtual screening combined with in vitro radiometric-based scintillation proximity assay (SPA). Substructure search based on the scaffold of the most active 9 afforded 26 additional analogues, and SPA results indicated that two analogues (9–1 and 9–2) showed increased PRMT5 inhibitory activity compared with the parental compound. Resynthesis of 9, 9–1, and 9–2 confirmed their PRMT5 enzymatic inhibition activity. In addition, compound 9–1 displayed selectivity against PRMT5 over other key homological members (PRMT1 and CARM1 (PRMT4)). While the structure–activity relationship (SAR) of this series of compounds was discussed to provide clues for further structure optimization, the probable binding modes of active compounds were also probed by molecular docking and molecular dynamics simulations. Finally, the antiproliferative effect of 9–1 on MV4-11 leukemia cell line was confirmed and its impact on regulating the target gene of PRMT5 was also validated. The hit compounds identified in this work have provided more novel scaffolds for future hit-to-lead optimization of small-molecule PRMT5 inhibitors.

Screening of the NIH Clinical Collection for inhibitors of HIV-1 integrase activity

Drug repurposing offers a validated approach to reduce drug attrition within the drug discovery and development pipeline through the application of known drugs and drug candidates to treat new indications. Full exploitation of this strategy necessitates the screening of a vast number of molecules against an extensive number of diseases of high burden or unmet need and the subsequent dissemination of the findings. In order to contribute to endeavours within this field, we screened the 727 compounds comprising the US National Institutes of Health (NIH) Clinical Collection through an HIV-1 (human immunodeficiency virus type 1) integrase stand transfer inhibition assay on an automated scintillation proximity assay platform. Only two compounds were identified within the initial screen, with cefixime trihydrate and epigallocatechin gallate found to reduce integrase strand transfer activity at IC50 values of 6.03±1.29 ?M and 9.57±1.62 ?M, respectively. However, both cefixime trihydrate and epigallocatechin gallate retained their low micromolar inhibitory activity when tested against a raltegravir-resistant integrase double mutant (FCIC50 values of 0.83 and 0.06, respectively), were ineffective in an orthogonal strand transfer ELISA (<30% inhibition at 100 ?M) and produced negligible selectivity index values (<1) in vitro. While no useful inhibitors of HIV-1 integrase strand transfer activity were found within the NIH Clinical Collection, the identification of two assay-disrupting molecules demonstrates the importance of consideration of non-specific inhibitors in drug repurposing screens.
 Significance: 
 
 This study is the first to screen the US NIH Clinical Collection for potential HIV-1 integrase inhibitors.
 The pervasive nature of promiscuous inhibitors is emphasised.

Discovery and Characterization of Chemical Compounds That Inhibit the Function of Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen, is highly susceptible to developing resistance to multiple antibiotics. The gene encoding aspartyl-tRNA synthetase (AspRS) from P. aeruginosa was cloned and the resulting protein characterized. AspRS was kinetically evaluated, and the KM values for aspartic acid, ATP, and tRNA were 170, 495, and 0.5 μM, respectively. AspRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 1690 chemical compounds, resulting in the identification of two inhibitory compounds, BT02A02 and BT02C05. The minimum inhibitory concentrations (MICs) were determined against nine clinically relevant bacterial strains, including efflux pump mutant and hypersensitive strains of P. aeruginosa. The compounds displayed broad-spectrum antibacterial activity and inhibited growth of the efflux and hypersensitive strains with MICs of 16 μg/mL. Growth of wild-type strains were unaffected, indicating that efflux was likely responsible for this lack of activity. BT02A02 did not inhibit growth of human cell cultures at any concentration. However, BT02C05 did inhibit human cell cultures with a cytotoxicity concentration (CC50) of 61.6 μg/mL. The compounds did not compete with either aspartic acid or ATP for binding AspRS, indicating that the mechanism of action of the compound occurs outside the active site of aminoacylation.

The design and utilization of the scintillation proximity assay to screen for p300 inhibitors

The design and utilization of the scintillation proximity assay to screen for p300 inhibitors