AlphaScreen Technology Research Articles

Aptamer-mediated nanoparticle interactions: from oligonucleotide-protein complexes to SELEX screens.

Aptamers are oligonucleotides displaying specific binding properties for a predetermined target. They can be easily immobilized on various surfaces such as nanoparticles. Functionalized particles can then be used to various aims. We took advantage of the AlphaScreen(®) technology for monitoring aptamer-mediated interactions. A particle bearing an aptamer contains a photosensitizer whereas another type of particle contains a chemiluminescer. Irradiation causes the formation of singlet oxygen species in the photosensitizer-containing bead that in turn activates the chemiluminescer. Luminescence emission can be observed if the two types of beads are in close proximity (<200 nm). This is achieved when the cognate ligand of the aptamer is grafted onto the chemiluminescer-containing bead. Using this technology we have screened oligonucleotide libraries and monitored aptamer-protein interactions. This constitutes the basis for aptamer-based analytical assays.

Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target.

Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)–compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni2+-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

Development of an HTS-Compatible Assay for the Discovery of ASK1 Signalosome Inhibitors Using AlphaScreen Technology

Genetic target validation studies have demonstrated that the apoptosis signal-regulating kinase 1 (ASK1) represents an important target for the treatment of rheumatoid arthritis, cardiac diseases, and several neurodegenerative disorders. To identify small-molecule inhibitors of ASK1, we have developed a high-throughput screening-compatible, homogenous, biochemical assay using AlphaScreen technology. This novel assay design utilizes purified stress-activated ASK1 signalosome complex, and it monitors phosphorylation of its full-length native substrate, MKK6. The assay has been optimized in a 384-well format and validated by screening the Sigma LOPAC library. The results presented here demonstrate that the assay is sensitive and robust with a Z' factor value of 0.88±0.04 and a signal-to-background ratio of 11, indicating that this assay can be used to screen large chemical libraries to discover novel inhibitors of ASK1.

Development of a High-Throughput AlphaScreen Assay for Modulators of Synapsin I Phosphorylation in Primary Neurons

Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders.

A Novel High Throughput Biochemical Assay to Evaluate the HuR Protein-RNA Complex Formation

The RNA binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the stabilization and translation of a number of mRNAs into the cytoplasm, dictating their fate. We applied the AlphaScreen technology using purified human HuR protein, expressed in a mammalian cell-based system, to characterize in vitro its binding performance towards a ssRNA probe whose sequence corresponds to the are present in TNFα 3’ untranslated region. We optimized the method to titrate ligands and analyzed the kinetic in saturation binding and time course experiments, including competition assays. The method revealed to be a successful tool for determination of HuR binding kinetic parameters in the nanomolar range, with calculated Kd of 2.5±0.60 nM, k on of 2.76±0.56*106 M-1 min-1, and k off of 0.007±0.005 min-1. We also tested the HuR-RNA complex formation by fluorescent probe-based RNA-EMSA. Moreover, in a 384-well plate format we obtained a Z-factor of 0.84 and an averaged coefficient of variation between controls of 8%, indicating that this biochemical assay fulfills criteria of robustness for a targeted screening approach. After a screening with 2000 small molecules and secondary verification with RNA-EMSA we identified mitoxantrone as an interfering compound with rHuR and TNFα probe complex formation. Notably, this tool has a large versatility and could be applied to other RNA Binding Proteins recognizing different RNA, DNA, or protein species. In addition, it opens new perspectives in the identification of small-molecule modulators of RNA binding proteins activity.

ELAVL1/HuR‐mediated accumulation of SQSTM1/p62 during proteasomal inhibition in human ARPE‐19 cells

AbstractPurpose Impaired protein degradation in retinal pigment epithelial (RPE) cells contributes to age‐related macular degeneration (AMD) pathogenesis. SQSTM1/p62 protein is involved in autophagy and proteasome‐mediated proteolysis, and expressed strongly under toxic stimuli. ELAVL1/HuR is a RNA‐binding protein which, in response to various stressors, regulates gene expression, finally affecting key cellular functions. Main aims of our study were to investigate whether p62 mRNA would be a target of HuR protein, and whether a link between these two proteins exists in RPE cells. Finally, the effects of proteasome inhibition and autophagy induction on p62 and HuR levels in RPE cells were also evaluated.Methods AlphaScreen technology and immunoprecipitation coupled with real‐time qPCR were used to study the binding between HuR protein and p62 mRNA. ARPE‐19 cells were treated with MG‐132 (proteasome inhibitor; 5µM) and/or AICAR (AICA ribonucleotide, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside; 2mM). P62 and HuR levels were analyzed by real‐time qPCR and Western blotting. HuR silenced ARPE‐19 cells and negative control cells were also employed.Results MG‐132 treatment up‐regulates HuR, which binds p62 mRNA thus contributing, at post‐transcriptional level, to the increase of p62 protein level in ARPE‐19 cells. The addition of AICAR promoted cleansing by autophagy of p62, but not HuR.Conclusion p62, whose mRNA represents a novel target of HuR, is positively regulated as a cellular response to proteasome inhibition. P62 is degraded by autophagy‐mediated pathway, while HuR through proteasome. These findings may be relevant for AMD.

Development of a High-Throughput Screening Assay for Inhibitors of Small Ubiquitin-Like Modifier Proteases

Small ubiquitin-like modifier (SUMO1–3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high-throughput screening (HTS)–compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z′ factor was 0.83 ± 0.04, confirming the suitability for HTS applications.

Development of a High-Throughput Screening–Compatible Assay for the Discovery of Inhibitors of the AF4-AF9 Interaction Using AlphaScreen Technology

Rearrangements of the mixed-lineage leukemia (MLL) gene occur predominately in pediatric leukemia cases and are generally predictors of a poor prognosis. These chromosomal rearrangements result in fusion of the protein MLL to one of more than 60 protein partners. MLL fusions are potent inducers of leukemia through activation of oncogene expression; therefore, targeting this transcriptional activation function may arrest MLL-rearranged (MLL-R) leukemia. Leukemic cell lines harboring the most common fusion protein, MLL-AF4, require the direct interaction of AF4 with the transcription factor AF9 to survive and self-renew; disrupting this interaction with a cell-penetrating AF4-derived peptide results in cell death, suggesting that the AF4-AF9 interaction could be a viable target for a novel MLL-R leukemia therapy. Here we describe the use of AlphaScreen technology to develop a high-throughput screening (HTS) assay to detect nonpeptidic inhibitors of AF4-AF9 binding. The assay is economical, requiring only low nanomolar concentrations of biotinylated AF4-derived peptide and FLAG-tagged AF9 in low-volume 384-well plates. A Z'-factor of 0.71 and a signal-to-background ratio of 21.3 showed the assay to be robust, and sensitivity to inhibition was demonstrated with competing AF4-derived peptides. Two pilot screens comprising 5,680 compounds served as validation for HTS at Nemours and the Broad Institute. Assay artifacts were excluded using a counterscreen comprising a biotinylated FLAG peptide. This is the first reported HTS-compatible assay to identify compounds that inhibit a key binding interaction of an MLL fusion partner, and the results presented here demonstrate suitability for screening large chemical libraries in high-density, low-volume plate formats.

Abstract 3337: Modulating the APE1/NPM1 interaction in cancer.

Abstract DNA repair pathways protect the genome from endogenous and environmental DNA damage. Such defensive mechanisms, however, enable tumor cells to survive DNA damage caused by chemo- and radio-therapy. Moreover, alterations of DNA repair pathways that may occur during tumorigenesis can make cancer cells reliant on a subset of repair enzymes for survival. Hence, knowledge of the mechanisms involved in the control of DNA repair proteins is of great interest for cancer diagnosis and treatment. The Apurinic/apyrimidinc endonuclease 1 (APE1) is an essential protein in eukaryotic cells. As the main AP-endonuclease in mammals, this protein is central to the base excision DNA repair pathway (BER). Beside its role in the maintenance of genomic stability, APE1 modulates gene expression by tuning the redox status of several transcription factors. Moreover, recent reports suggest that APE1 may take part in RNA processing and degradation, thus controlling the transcriptional output of the cell. Overexpression and aberrant localization of APE1 are recurrent phenotypes in tumors. Both situations are prognostic indicators of aggressiveness and onset of resistance. Interestingly, downregulation or inhibition of APE1 sensitizes cells to pharmacologically relevant gentoxins. We identified and characterized the molecular association between APE1 and Nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability, rRNA maturation and chromatin remodeling. The association with NPM1 modulates subcellular localization and endonuclease activity of APE1. Accordingly, NPM1−/− cells show lower BER capacity, and expression of an APE1 mutant unable to interact with NPM1 severely impairs cell proliferation. We recently reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, along with a poorer prognosis for patients having higher NPM1 levels. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Targeting the APE1/NPM1 interaction therefore represents a novel strategy for the development of anticancer drugs, as tumor cells which rely on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than normal cells. We exploited the AlphaScreen technology to screen for small molecules that act as inhibitors of the APE1/NPM1 interaction. Our approach detected a set of compounds able to interfere with the APE1/NPM1 association in vivo. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to genotoxins. To the best of our knowledge, this is the first attempt to target the APE1 interactome. Given the prognostic significance of the APE1 and NPM1 overexpression, these compounds might prove particularly effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas. Citation Format: Mattia Poletto, Dorjbal Dorjsuren, Lisa Lirussi, Carlo Vascotto, Pasqualina L. Scognamiglio, Daniela Marasco, Ajit Jadhav, David J. Maloney, Anton Simeonov, David M. Wilson, Gianluca Tell. Modulating the APE1/NPM1 interaction in cancer. [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 3337. doi:10.1158/1538-7445.AM2013-3337

Establishment of a robust dengue virus NS3–NS5 binding assay for identification of protein–protein interaction inhibitors

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3–NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z′ factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins.

Abstract 2806: BRAF and MEK inhibitor profiling across 240 tumor cell lines to correlate with sensitivity and resistant biomarkers

Abstract Activated kinases that induce cell proliferation have been attractive targets for targeted cancer therapy development. Cancer cells could become dependent on tumor-specific activated kinases and this tendency has been coined oncogene addiction. Here, we investigated three activated kinase inhibitors: BRAF inhibitor, PLX-4032 analog, and the MEK inhibitors, CI1040 and PD0325901. We established a high throughput cellular approach to profile 240 human tumor cell lines identifying genotype-correlated sensitivity or resistance. Proliferative, apoptotic and cell cycle arrest responses were measured using multiplexed high content screening with automated fluorescence microscopy and image analysis based technology. Growth index was measured using nuclear dye. Activated caspase 3 antibodies were used for the apoptosis induction detection. Phospho-histone H3 antibodies were used to measure the cell cycle block. We generated cell line profiles to reveal drug sensitivity and resistance patterns that may correlate with the clinical genotype responses. Cell lines with BRAFV600E mutations showed overlapping sensitivity to all three MEK and BRAF inhibitors. RAS mutations confer resistance to the BRAF inhibitor and confer sensitivity to both MEK inhibitors. In addition, we used Alphascreen technology to measure phosphoERK across the 240 non-treated tumor cell line panel. We found that the majority of PLX-4032 sensitive cell lines expressed high levels of phosphoERK. In addition, we investigated BRAF, MEK and EGFR inhibitor combinations to evaluate potential synergies. This preclinical approach can be used to predict mechanisms of susceptibility or resistance to these agents which in turn could be used for the optimization of targeted cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2806. doi:1538-7445.AM2012-2806

Measurement of cAMP in an Undergraduate Teaching Laboratory, Using ALPHAscreen Technology

Adenosine 3',5'-monophosphate (cAMP) is a cellular second messenger with central relevance to pharmacology, cell biology, and biochemistry teaching programs. cAMP is produced from adenosine triphosphate by adenylate cyclase, and its production is reduced or enhanced upon activation of many G protein-coupled receptors. Therefore, the measurement of cAMP serves as an indicator of receptor activity. Although there are many assays available for measuring cAMP, few are suitable for large class teaching, and even fewer seem to have been adapted for this purpose. Here, we describe the use of bead-based ALPHAscreen (Amplified Luminescent Proximity Homogenous Assay) technology for teaching a class of more than 300 students the practical aspects of detecting signal transduction. This technology is applicable to the measurement of many different signaling pathways. This resource is designed to provide a practical guide for instructors and a useful model for developing other classes using similar technologies.

Abstract B99: Targeting TPR cochaperones of the Hsp90 molecular chaperone complex for specific inhibition.

Abstract The Hsp90 molecular chaperone complex plays an important role in tumour biology by aiding oncogenic client proteins in their stability and activity. Targeted therapeutics that disrupt the Hsp90 function have shown promise in clinical trials (eg 17-allylamino-17-demethoxygeldanamycin, 17-AAG, and the synthetic resorcinylic isoxazole NVP-AUY922). These drugs bind Hsp90's ATP/ADP-site, thereby inhibiting the ATP-coupled conformational cycle which plays a crucial part of chaperoning several oncogenic client proteins. Hsp90 functions as part of a chaperone complex that requires the involvement of a diverse series of cochaperones, including the Hsp70/Hsp90 organising protein (Hop). These proteins perform regulatory roles or act as scaffolds to ensure complex stability and correct client selection. A subset of structurally related but functionally diverse cochaperones interact with the C-terminal MEEVD sequence of Hsp90 through a tetratricopeptide repeat (TPR) domain. This work aims to discover inhibitors of this interaction to allow selective inhibition of Hsp90 functions and provide chemical probes for studying cochaperone biology. Two biochemical binding assays were developed for screening compounds for inhibition of the Hsp90-TPR interaction. The assays used AlphaScreen and LANCE technology and provided a high-throughput platform for assessing compounds for inhibition against a panel of TPR proteins. Hit compounds were validated using biophysical techniques. Inhibitor effects on TPR stability in the absence of a competing substrate were assessed using a thermal shift assay. In addition, ligand binding sites were identified using 1H-15N HSQC chemical shift mapping onto an existing crystal structure. These robust assays allowed high-throughput screening of an 80,000 compound library with mean Z'=0.92 and CV=2.6%, giving a hit rate of 1.3%. In addition compounds identified through in silico screening methods have also been tested using these assays. Three general inhibitor chemotypes have been discovered. Chemical shift mapping has shown that although these inhibitors are weak, it is likely that several different binding sites are being exploited. Improvements in potency are being pursued through analogue by catalogue methods guided by the NMR shift mapping data. Specific Hsp90-TPR inhibitors will provide important tools for investigating Hsp90/cochaperone complexes and the dependency of client subsets on particular cochaperones. In addition, inhibitors of some TPR cochaperones may sensitize cells to existing Hsp90 inhibitors or perhaps act as independent inhibitors of specific Hsp90 functions. This work has discovered several weak inhibitors of this interaction in a biochemical setting and characterised their interactions with Hop TPR 2A. Further optimisation could provide useful chemical probes and potential starting points for drug discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B99.

Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gα(o)-RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gα(o)-RGS17 protein-protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC(50) <10 µM in dose-response experiments. Four exhibited IC(50) values <6 µM while inhibiting the Gα(o)-RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

Abstract 1375: A homogeneous high-throughput screening assay for inhibitors of the interaction of MLL-AF4 and AF9 in pediatric leukemia

Abstract Chromosomal translocations of the mixed lineage leukemia (MLL) gene occur in 5-10% of pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), including up to 80% of infant ALL and most treatment-related secondary AML. These MLL rearrangements result in a portion of the MLL protein being fused to one of a number of transcription factors. The prognosis for MLL-rearranged (MLL-R) leukemia is much poorer than for leukemia devoid of MLL fusions; five-year survival of &amp;lt;40% in the case of infant ALL. MLL fusions are potent inducers of leukemia, through recruitment of a protein complex that activates transcription of the HOX (homeobox) oncogene HOXA9, which in turn blocks normal differentiation of progenitor cells and confers a stem cell-like phenotype. The transcription factor AF4 is the most common MLL fusion partner, and MLL-AF4 is associated with a particularly poor prognosis. Direct interaction of MLL-AF4 with the transcription factor AF9 has been shown to be critical to leukemic transformation. Here we describe the development of a homogeneous high-throughput screening (HTS) assay for small-molecule inhibitors of the binding of MLL-AF4 to AF9 and the validation of this assay in pilot HTS. A recent study showed that peptides containing as few as 10 amino acids derived from the AF9 binding site in AF4 bind to AF9 with low nanomolar potency and selectively kill MLL-AF4-containing leukemia cell lines. We have developed an assay that measures binding of an AF4-derived peptide to AF9 protein using AlphaScreen technology, a homogeneous proximity-based method well suited to HTS in 384- and 1536-well plates. Laser excitation of donor beads results in the generation of singlet oxygen, which encounters acceptor beads brought into close proximity by the binding of AF4 peptide to AF9, giving an amplified luminescent signal. Inhibitors are identified by reduction of the luminescent signal. We have validated the assay by demonstrating low nanomolar inhibition by several AF4-derived peptides and by screening a library of 2000 drugs and natural products. The next step is transfer of the assay to the Broad Institute (Cambridge, MA) for HTS against the 400,000-compound NIH library. The ultimate goal is the optimization of lead compounds as novel therapies targeted to pediatric leukemia harboring MLL-AF4, both as stand-alone agents and as synergistic potentiators of existing chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2011-1375

Molecular description of non-autoimmune hyperthyroidism at a neonate caused by a new thyrotropin receptor germline mutation

BackgroundConstitutively activating germline mutations in the thyrotropin receptor (TSHR) gene result in non-autoimmune hyperthyroidism and can be transmitted as a dominant trait or occur sporadically. These mutations are mostly located in the serpentine part of this G-protein coupled receptor.MethodsSequencing exon 9 and 10 of the thyrotropin receptor gene in a two months old patient identified a mutation which was functionally characterized after transient transfection into COS-7 cells. Cell surface localization was investigated by an ELISA approach and for signalling properties we measured cAMP by alpha screen technology for Gs/adenylyl cyclase activation and use a reporter gene assay for determination of Gq/11 phospholipase C-β activation.ResultsWe detected a heterozygous mutation in the first extracellular loop of the TSHR gene leading to an exchange of an isoleucine residue for asparagine at amino acid position 486 (I486N). Cell surface localization was reduced to 51% of wild-type TSHR. Functional characterization of the mutant receptor revealed constitutive activation of the Gs/adenylyl cyclase pathway, in contrast basal activity of the Gq/11 pathway was comparable to the wild-type. The bovine TSH-induced cAMP accumulation was slightly reduced, but IP3 signaling was impaired.ConclusionWe identified a new TSHR germline mutation (I486N) in a neonate with non-autoimmune sporadic hyperthyroidism. The mutation is located at the extracellular loop 1 and exhibits an increase in basal cAMP accumulation, but unexpectedly impairs the capability for TSH induced Gq mediated signaling. The TSHR homology model suggests isoleucine 486 as a potential key-player for induction of signal transduction by an interplay with further activation sensitive extracellular parts.

Abstract 2681: EWS-FLI1 as a molecular target: Small molecule inhibitors for a disordered protein

Abstract Ewing's sarcoma family of tumors are characterized by the EWS-FLI1 oncogenic fusion protein. Our previous studies show that RNA Helicase A (RHA) enhances EWS-FLI1 driven oncogenesis, and interruption of this protein-protein complex by small molecule inhibitors validate this interaction as a unique therapeutic target (NatMed 2009 Jul;15(7):750-6). EWS-FLI1 is a significantly hydrophobic disordered protein with unknown three-dimensional structure, which therefore precludes standard structure-based small molecule design. Given the challenges of drug design targeted to EWS-FLI1, we hypothesize that the optimization of a peptide displacement assay using small molecules can predict and direct the design of more potent analogs. Fluorescence polarization (FP) was originally used to measure RHA peptide displacement from EWS-FLI1, but is time consuming and reagent limiting. To identify new strategies for higher-throughput screening, a TECAN ULTRA 384 was used for assay detection in a 96-well plate format. However, this resulted in high background and low signal. A second approach, AlphaScreen (amplified luminescent proximity homogenous assay, PerkinElmer), uses a bead-based interaction system based on energy conversion and generation of chemiluminescence when acceptor and donor beads are brought together during binding. Results show a 6-fold dynamic range between bound and quenched beads, in comparison to just 2.5-fold for FP and less than two-fold for TECAN. Our small molecule, YK-4-279, is able to reduce the signal down to baseline, indicating that YK-4-279 dissociates the RHA peptide and EWS-FLI1. Key negative controls support specific binding between EWS-FLI1 and the RHA peptide. In addition, YK-4-279 was unable to quench the signal from a positive control using immunoglobulin, supporting the conclusion that YK-4-279 disrupts the specific protein-peptide binding rather than directly extinguishing the chemiluminescence. Data from the AlphaScreen will be used as input to a quantitative-structure activity relationship (QSAR) method. This QSAR method will predict biological activity from chemical structure and the knowledge gained from AlphaScreen will aid in the optimization and design of future analogs. In our initial work, we used the Free-Wilson QSAR method, converting the structure of the small molecules into a numerical format allowing for a mathematical comparison between structures, to populate a table with 32 small molecule analogs of YK-4-279. The AlphaScreen data may also be used with other QSAR methods, to which we may transition in later work. AlphaScreen technology provides a method to quantitatively compare binding of small molecules in a high-throughput screening system, yields a greater dynamic range with more specificity, and provides data that will help to predict the binding of other analogs with similar chemical structures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2681.

Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design

Author SummaryDepression is the most common of psychiatric illnesses, with prevalence estimates ranging from 5% to 20% within the general population. The design of effective treatments for this disorder is a challenging process, and the use of antidepressants has an overall low clinical efficacy as full remission only occurs in one-third of the patients. Moreover, the time between initial treatment and beneficial effects is relatively protracted. These limitations confirm the need to find new biological targets and drugs for the treatment of depression. We recently identified a conserved mouse potassium channel protein called TREK-1 (KCNK2) as a new target for treating depression. Here, we demonstrate that spadin, a natural peptide derived from a propeptide released in blood, is able to block the TREK-1 channel activity and has an antidepressant effect in mouse models of depression. We showed that spadin is an efficient antidepressant in mice that acts much faster (4 d versus several weeks) than fluoxetine, the most commonly used antidepressant. Our results with spadin in mice highlight the potential for novel and more efficacious treatments for depression in humans.

Identification of a small-molecule inhibitor of the interaction between Survivin and Smac/DIABLO

The protein Survivin is selectively overexpressed in a variety of cancers, but not in normal tissues. It has been reported to be involved in cell survival and cell division. However, the molecular mechanisms involved in its function are not clear, although several binding partner proteins have been proposed to date. Here, we report the identification of a novel small molecule Survivin antagonist, which disrupts the Survivin–Smac/DIABLO interaction in cells. In order to identify Survivin-directed antagonists, we developed a high-throughput screening system based on AlphaScreen technology, which allows the identification of small molecules with the ability to inhibit the interaction of Survivin with Smac/DIABLO or INCENP in vitro. We screened chemical libraries, generated in-house, using this system and identified a 5-deazaflavin analog (compound 1) as a hit compound that selectively inhibited the interaction of Survivin with Smac/DIABLO but not INCENP. In cultured cells, compound 1 abrogated the formation of the complex between Survivin and Smac/DIABLO. In addition, this compound was able to sensitize cultured cells to doxorubicin-mediated DNA damage stress and synergistically enhance apoptotic cell death. Thus, the small-molecule inhibitor described here may serve as a proof-of-principle agent for discriminating between the multiple functions of Survivin.