Experimental Therapeutics Center

Nanos, Singapore

Experimental Therapeutics Center

Nanos, Singapore
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Huang H.,National University of Singapore | Tan B.,National University of Singapore | Shen Y.,National University of Singapore | Tao J.,National University of Singapore | And 11 more authors.
Neuron | Year: 2012

Adenosine-to-inosine RNA editing is crucial for generating molecular diversity, and serves to regulate protein function through recoding of genomic information. Here, we discover editing within Ca V1.3 Ca 2+ channels, renown for low-voltage Ca 2+-influx and neuronal pacemaking. Significantly, editing occurs within the channel's IQ domain, a calmodulin-binding site mediating inhibitory Ca 2+-feedback (CDI) on channels. The editing turns out to require RNA adenosine deaminase ADAR2, whose variable activity could underlie a spatially diverse pattern of Ca V1.3 editing seen across the brain. Edited Ca V1.3 protein is detected both in brain tissue and within the surface membrane of primary neurons. Functionally, edited Ca V1.3 channels exhibit strong reduction of CDI; in particular, neurons within the suprachiasmatic nucleus show diminished CDI, with higher frequencies of repetitive action-potential and calcium-spike activity, in wild-type versus ADAR2 knockout mice. Our study reveals a mechanism for fine-tuning Ca V1.3 channel properties in CNS, which likely impacts a broad spectrum of neurobiological functions. Huang et al. find that RNA editing of the voltage-gated calcium channel Cav1.3 IQ domain acts as a regulatory mechanism for calcium-dependent channel inactivation. Given widespread roles for Cav1.3 channels invivo, this mechanism may have consequences for a broad range of physiological functions. © 2012 Elsevier Inc.

Proffitt K.D.,National University of Singapore | Madan B.,National University of Singapore | Ke Z.,Experimental Therapeutics Center | Pendharkar V.,Experimental Therapeutics Center | And 4 more authors.
Cancer Research | Year: 2013

Porcupine (PORCN) is a membrane bound O-acyltransferase that is required for Wnt palmitoylation, secretion, and biologic activity. All evaluable human Wnts require PORCN for their activity, suggesting that inhibition of PORCN could be an effective treatment for cancers dependent on excess Wnt activity. In this study, we evaluated the PORCN inhibitor Wnt-C59 (C59), to determine its activity and toxicity in cultured cells and mice. C59 inhibits PORCN activity in vitro at nanomolar concentrations, as assessed by inhibition of Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of β-catenin reporter activity. In mice, C59 displayed good bioavailability, as once daily oral administration was sufficient to maintain blood concentrations well above the IC50. C59 blocked progression of mammary tumors in MMTV-WNT1 transgenic mice while downregulating Wnt/β-catenin target genes. Surprisingly, mice exhibit no apparent toxicity, such that at a therapeutically effective dose there were no pathologic changes in the gut or other tissues. These results offer preclinical proof-of-concept that inhibiting mammalian Wnts can be achieved by targeting PORCN with small-molecule inhibitors such as C59, and that this is a safe and feasible strategy in vivo. © 2012 AACR.

News Article | November 30, 2016
Site: www.eurekalert.org

An international group of researchers report success in mice of a method of using positron emission tomography (PET) scans to track, in real time, an antibody targeting a hormone receptor pathway specifically involved in prostate cancer. This androgen receptor pathway drives development and progression of the vast majority of prostate cancers. The technique shows promise, the investigators say, as a novel way to use such an antibody to detect and monitor prostate and other hormone-sensitive cancers, as well as to guide therapy in real time. "The findings show that individually tailored imaging agents can provide a unique way of looking at disease progression in real time and in a noninvasive manner," says Daniel Thorek, Ph.D., assistant professor of radiology and radiological science at the Johns Hopkins University School of Medicine, and the paper's first author. "Perhaps someday we can put a personalized antibody such as the one we created in our study on a therapeutic agent and conduct cancer treatment using imaging with very high specificity." A summary of the findings was published in Science Translational Medicine on Nov. 30. Thorek says the success is especially important given the challenges of working with small-animal models. "We managed to very accurately and precisely monitor the mouse prostate, and that leads us to hope that a similar approach can be used to guide treatment in people," he adds. Current clinical practice detects prostate cancer by tracking the androgen receptor pathway -- a marker for the cancer -- by testing blood for prostate-specific antigen (PSA). Presence of elevated PSA indicates that the androgen receptor pathway is active and may indicate prostate cancer is present. PSA concentration in the blood, however, is affected by numerous factors, such as age and type of tumor, making it difficult to determine true androgen receptor pathway activation. Furthermore, attempts to target PSA with an antibody is complicated by the "washing out" of the antibody-PSA complex, a process in which the complex is formed but does not remain near the disease site, thus making it difficult to definitively identify and measure disease sites. In the new study, the investigators developed a new antibody called 11B6 to target human kallikrein-related peptidase 2 (hK2), another antigen that indicates androgen receptor pathway activation. Unlike PSA, hK2 is specifically active only in the prostate and an aggressive type of breast cancer. By binding free, unbound hK2 to 11B6, the research team found that the newly formed 11B6-hK2 complex is taken directly back into the cancerous cell rather than washed out. This biological process relies on a transport mechanism involving the neonatal Fc receptor, in which cells are able to recognize and take in antibodies. The Fc receptor is most well-known for how antibodies in mothers' milk are able to pass from the gut of the baby into newborns' bloodstream to provide them with immunity. As far as the research team is aware, this is the first study to exploit the biological mechanism for imaging purposes, Thorek says. In the next phase of their experiments, the team made 11B6 "light up" during PET and fluorescence imaging by binding it to zirconium-89, creating a traceable radiochemical compound called 89Zr-11B6. By imaging the 89Zr-11B6 using PET, the team showed that binding 11B6 to hK2 can measure activity of cancerous lesions robustly, in both soft tissue and bone. Prostate and breast cancer often metastasize to bone, therefore detection of lesions in all areas of the body is critical. To further demonstrate the potential value of 89Zr-11B6 imaging, the team tested the imaging agent in disease models under standard treatment regimens. In one such case, disease activity was imaged and quantified in mice treated with saline and a second group with enzalutamide, a drug used to treat prostate cancer by inhibiting the androgen receptor hormone activity. All of the mice had prostate cancer. Following initial castration, imaging of 89Zr-11B6 allowed the research team to see lower androgen receptor pathway activity, as one might expect. This effect was augmented in the animals with adjuvant enzalutamide treatment. This may inform current clinical practice as the use of adjuvant enzalutamide after castration may show benefit to patients with prostate cancer. By tracking the antibody localization to disease sites in real time, the team hopes this may be a way to determine optimal dosages that don't compromise efficacy while avoiding negative side effects. Imaging of the antibody uptake before and after treatment could, in theory, aid in the decision of whether to keep a patient on chosen drug. If a response is seen, the imaging agent could be used to choose the right dose, balancing the therapeutic effect and minimizing adverse effects. And if there is not an imaging change with a particular drug, this tool would provide a caregiver with rapid information to discontinue ineffective treatments, saving time and cost. The team is currently conducting preliminary nonhuman primate toxicity tests, the final step before applying for human clinical trials, and has thus far found no adverse effects. Other authors on this paper include Diane S. Abou and Marise R.H. van Voss of the Johns Hopkins University School of Medicine; Philip A. Watson, Sang-Gyu Lee, Anson T. Ku, Kwanghee Kim, Michael G. Doran, Elmer Santos, Darren Veach, Mesruh Turkekul, Emily Casey, Jason S. Lewis, Howard I. Scher, Hans Lilja, Steven M. Larson and David Ulmert of Memorial Sloan Kettering Cancer Center; Stylianos Bournazos of Rockefeller University; Katharina Braun of the University of Bochum; Kjell Sjöström of Innovagen AB; Urpo Lamminmäki of the University of Turku; Sven-Erik Strand of Lund University; and Mary L. Alpaugh of Rowan University. Funding for this study was provided by the National Cancer Institute of the National Institutes of Health (P30 CA008748, P30 CA006973, P30 CA008748-48, S10 RR020892-01, S10 RR028889-01, R33 CA127768-02, P50-CA86438), the National Institutes of Health Molecular Imaging Fellowship Program (5R25CA096945-07), the Geoffrey Beene Cancer Research Center, the W.H. Goodwin and A. Goodwin and their Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center, the Radiochemistry and Molecular Imaging Probe Core (P50-CA086438), the Steve Wynn Prostate Cancer Foundation Young Investigator Award, the Knut and Alice Wallenberg Foundation, the Bertha Kamprad Foundation and the David H. Koch Fund of the Prostate Cancer Foundation, the Ludwig Center for Cancer Immunotherapy, the Swedish Cancer Society, the Swedish National Health Foundation, the Swedish Research Council (Medicine- 20095), the Memorial Sloan Kettering Cancer Center Specialized Programs of Research Excellence in Prostate Cancer (P50 CA92629), the Sidney Kimmel Center for Prostate and Urologic Cancers and the Hascoe Charitable Foundation. D.L.J.T., D.U., U.L., S.-E.S., and H.L. are shareholders of Diaprost Inc. D.L.J.T., S.-E.S., and D.U. currently serve as board members of Diaprost Inc. D.L.J.T., A.K., S.-E.S., S.M.L., and D.U. are inventors on a patent (62257179) submitted by the MSKCC that covers systems, methods and compositions for imaging AR axis activity in carcinoma. D.U. is also the inventor on a patent (20060182682) held by Diaprost Inc. that covers diagnostic imaging of PCa using 11B6. S.-E.S. and U.L. are inventors on a patent application (WO2015075445) submitted by Diaprost Inc. that covers the humanized anti-hK2 antibody.

Wong J.S.,P53 Laboratory | Wong J.S.,A-Life Medical | Warbrick E.,A-Life Medical | Vojtesk B.,Masaryk Memorial Cancer Institute | And 2 more authors.
Oncotarget | Year: 2013

c-Met is a tyrosine receptor kinase which is activated by its ligand, the hepatocyte growth factor. Activation of c-Met leads to a wide spectrum of biological activities such as motility, angiogenesis, morphogenesis, cell survival and cell regeneration. c-Met is abnormally activated in many tumour types. Aberrant c-Met activation was found to induce tumour development, tumour cell migration and invasion, and the worst and final step in cancer progression, metastasis. In addition, c-Met activation in cells was also shown to confer resistance to apoptosis induced by UV damage or chemotherapeutic drugs. This study describes the development of monoclonal antibodies against c-Met as therapeutic molecules in cancer treatment/diagnostics. A panel of c-Met monoclonal antibodies was developed and characterised by epitope mapping, Western blotting, immunoprecipitation, agonist/antagonist effect in cell scatter assays and for their ability to recognise native c-Met by flow cytometry. We refer to these antibodies as Specifically Engaging Extracellular c-Met (seeMet). seeMet 2 and 13 bound strongly to native c-Met in flow cytometry and reduced SNU-5 cell growth. Interestingly, seeMet 2 binding was strongly reduced at 4°C when compared to 37°C. Detail mapping of the seeMet 2 epitope indicated a cryptic binding site hidden within the c-Met a-chain.

Arslan Yildiz A.,Institute of Materials Research and Engineering of Singapore | Kang C.,Experimental Therapeutics Center | Sinner E.-K.,University of Natural Resources and Life Sciences, Vienna
Analyst | Year: 2013

The hERG (human ether-à-go-go-related gene) potassium channel has been extensively studied by both academia and industry because of its relation to inherited or drug-induced long QT syndrome (LQTS). Unpredicted hERG and drug interaction affecting channel activity is of main concern for drug discovery. Although there are several methods to test hERG and drug interaction, it is still necessary to develop some efficient and economic ways to probe hERG and drug interactions. To contribute this aim, we have developed a biomimetic lipid membrane platform into which the hERG channel can be folded. Expression and integration of the hERG channel was achieved using a cell-free (CF) expression system. The folding of hERG in the biomimetic membrane system was investigated using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) and Imaging Surface Plasmon Resonance (iSPR). In addition, the hERG channel folded into our biomimetic membrane platform was used for probing the channel and drug interactions through fluorescence polarization (FP) assay. Our results suggest that the biomimetic system employed is capable of detecting the interaction between hERG and different channel blockers at varied concentrations. We believe that our current approach could be applied to other membrane proteins for drug screening or other protein-related interactions. © 2013 The Royal Society of Chemistry.

Roux K.J.,University of South Dakota | Roux K.J.,Florida College | Kim D.I.,University of South Dakota | Kim D.I.,Florida College | And 2 more authors.
Journal of Cell Biology | Year: 2012

We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximitydependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximitydependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. © 2012 Roux et al.

Keller T.H.,Experimental Therapeutics Center | Shi P.-Y.,Novartis | Wang Q.-Y.,Novartis
Current Opinion in Chemical Biology | Year: 2011

In an era of emerging and reemerging infectious diseases, and increasing multidrug resistance, the need to identify novel therapy is imperative. Unfortunately, the recent shift of the drug discovery paradigm from cellular screening to target-based approaches has not delivered the anticipated benefits. A recent renaissance of the traditional cell-based approach, on the other hand, has yielded several clinical candidates. Three successful examples are illustrated in this review, namely spiroindolone, thiazolidinone, and diarylquinoline for the treatment of malaria, hepatitis C virus, and tuberculosis, respectively. We describe in detail their identification, mechanism of action (MoA), and common features in the chemical structures. The challenges of the cell-based approach for anti-infective drug discovery are also discussed. We propose a shift from standard libraries to synthetic natural-product-like compound collections to improve the success of phenotypic lead finding and to facilitate the validation of hits. © 2011 Elsevier Ltd.

Say E.,sGSK Group | Tay H.-G.,Singapore Institute of Medical Biology | Zhao Z.-s.,sGSK Group | Baskaran Y.,sGSK Group | And 4 more authors.
Molecular Cell | Year: 2010

Loss of fragile X mental retardation protein FMR1 is the most common genetic cause of mental deficiency in man. We find that both FMR1 and the related FXR1 serve as direct binding partners for the Cdc42 effector PAK1. This involves an 11 residue segment in the PAK1 autoinhibitory domain that is exposed upon kinase activation and binds the FXR1 KH2 domain. Active PAK1 can phosphorylate FXR1 at Ser420; antibodies to this site show increased phosphorylation when fragile X proteins are recruited to stress granules. During zebrafish muscle development, FXR1 Ser420 phosphorylation is needed for protein function. The familial FMR1(I304N) mutation is biologically inactive, and FXR1(I304N) fails to bind PAK1. A different PAK1 binding-deficient mutant, FXR1(Q348K/E352A), fails to rescue loss of Zf-FXR1 unless combined with a gain-of-function S420D phosphomimetic. This is the first documented protein partner for the KH(2) domain of FMR1 or FXR1, and it has several implications for signaling by fragile X proteins. © 2010 Elsevier Inc. All rights reserved.

Lefave C.V.,Sloan Kettering Cancer Center | Squatrito M.,Brain Tumor Center | Vorlova S.,Sloan Kettering Cancer Center | Rocco G.L.,Sloan Kettering Cancer Center | And 6 more authors.
EMBO Journal | Year: 2011

In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-Î ±/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur dĝ€2Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16-or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness. © 2011 European Molecular Biology Organization | All Rights Reserved.

Poulsen A.,Experimental Therapeutics Center | Kang C.,Experimental Therapeutics Center | Keller T.H.,Experimental Therapeutics Center
Current Pharmaceutical Design | Year: 2014

Even though a number of groups have identified peptidic inhibitors for DENV and WNV proteases, and several high throughput screening campaigns have been performed, the progress towards drug candidates has been very slow. This is in stark contrast to the related NS3/NS4A protease of HCV for which two peptidomimetic drugs were approved in 2011. In this review we will compare the NS3 proteases of the flaviviruses WNV and DENV with that of HCV, and answer the question whether the flavivirus proteases are inherently more challenging, or whether the lack of success is simply due to the limited resources that have so far been invested in these neglected disease targets. © 2014 Bentham Science Publishers.

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