High Throughput Approaches

 

At the department of Pharmacology at UNC we use high-throughput technologies to discover and validate druggable targets as well as small molecule modulators of druggable targets.  Many of the approaches use high-thoughput screening (HTS) approaches which are facilitated by robotics and other automation strategies (see video).  Ultimately we hope to create novel treatments for human disease.
 
Emanuele Network Anal and Hi ThroughputHahn High ThroughputRoth High Throughput
Ubiquitin signaling networks Sensitive assays for high content screening Chemical biology and the receptorome

 

 

 Research:

 

Systems biology approaches to decode ubiquitin signaling networks

The cellular protein landscape is dynamically regulated through changes in transcription and by the ubiquitin dependent degradation of specific proteins. In addition to its critical role in regulated protein degradation, the ubiquitin pathway has recently been shown to play a more complex role in regulating signal transduction, through controlling protein interactions and localization. The diversity of ubiquitin signaling outputs, and the perturbation of the ubiquitin proteasome system (UPS) in cancer underscore the importance of understanding key ubiquitin signaling events. However, a key challenge in the ubiquitin field has been to connect UPS enzymes with the substrates that they regulate. Our lab applies emerging genetic and proteomic technologies to systematically explore ubiquitin dependent signal transduction during cell cycle progression and in response to DNA damage.  We are implementing and developing technologies that can assess global, proteome wide controlled by ubiquitination. Global Protein Stability Profiling (GPS) is a genetic platform that utilizes fluorescent reporters together with cell sorting to assess changes in protein stability. The GPS system employs a collection of more than 15,000 human open reading frames (ORFs) expressed from a fluorescent reporter construct to simultaneously assess changes in the stability of 15,000 human proteins. As a complement to GPS, we utilize a proteomic approach termed QUAINT (Quantitative Ubiquitylation Interrogation). QUAINT is a mass spectrometry based platform that quantitatively measures changes in protein ubiquitylation for endogenous proteins. Together, these emerging technologies will provide a deep snap shot in the regulated proteome and allow us to better understand global ubiquitin signaling networks regulated during cell growth, in response to stress and in during disease. 

Emanuele Network Anal and Hi Throughput

Michael Emanuele, PhD |Webpage | Email | Publications

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Sensitive assays for high content screening and live cell analysis

The image shows a novel merocyanine dye placed where a shift in the position of a charged  amino acid affects a heteroatom on the dye, leading to a large increase in dye intensity. Proteins labeled with very bright, long wavelength dyes are used in both drug screening assays and to study protein conformation in vivo.

We have developed novel fluorescent dyes and labeling approaches for high content screening. The dyes are attached to proteins where their spectrum changes in response to protein phosphorylation, conformational changes or ligand binding. They have proven valuable in drug discovery, generating bright signals for sensitive screening assays. In addition, membrane permeable derivatives of the dyes are being attached to drug molecules to examine their metabolism and fate within living cells. Current work is focused on understanding the photophysical properties of dyes to build reporters of specific protein functions, reporting endogenous protein activity, and the ability to trace multiple different protein activities in the same assay or cell. In vivo labeling approaches are being harnessed to use dye-based biosensors in high content screening. Another side to this work is the generation of biosensors via high throughput screening, using libraries of engineered scaffolds. Library members binding to the activated conformation of targets are modified to generate biosensors of endogenous signaling proteins.
 

Hahn High Throughput

Klaus M. Hahn, PhD | Webpage | Email| Publications


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Chemical Biology and the Receptorome


We have pioneered the approach of massively-parallel physical screening of the GPCR-ome. Our approach differs from conventional high-throughput (HTS) approaches in that we screen, in a parallel fashion, entire families of receptors simultaneously to discover molecular targets of biologically important molecules (peptides, drugs, natural products). This work is facilitated by the NIMH Psychoactive Drug Screening Program which is housed in the Roth lab.

Roth High Throughput 

Bryan Roth, MD, PhD |Webpage | Email | Publications

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People:

Nancy AllbrittonNancy Allbritton
Henrik Dohlman, PhD Henrik Dohlman
Michael Emanuele Michael Emanuele
Klaus M. Hahn, PhD Klaus Hahn
Gary Johnson Gary Johnson
Bryan Roth, MD, PhD Bryan Roth

Publications:


Ly T, Ahmad Y, Shlien A, Soroka D, Mills A, Emanuele MJ, Stratton MR, Lamond AI. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells. eLife 2014;3:e01630.

Olive AJ, Haff MG, Emanuele MJ, Sack LM, Barker JR., Elledge SJ, Starnbach MN. Chlamydia trachomatis-Induced Alterations in the Host Cell Proteome Are Required for Intracellular Growth. Cell Host Microbe. 2014 Jan 15;15(1):113-24.

Emanuele MJ, Elia EH, Xu Q, Thoma CR, Izhar L, Guo A, Rush J, Hsu PW, Yen HS, Elledge SJ. Global Identification of Modular Cullin-Ring Ligase Substrates. Cell. 2011 Oct 14;147(2):459-74. Epub 2011 Sep 29.

Emanuele MJ, Ciccia A, Elia AE, Elledge SJ. Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate. PNAS 2011. 108 (24) 9845-9850; Epub ahead of print May 31, 2011.
 
Konze, K.D., Ma, A., Li, F., Barsyte-Lovejoy, D., Parton, T., Macnevin, C.J., Liu, F., Gao, C., Huang, X.P., Kuznetsova, E., Rougie, M., Jiang, A., Pattenden, S.G., Norris, J.L., James, L.I., Roth, B.L., Brown, P.J., Frye, S.V., Arrowsmith, C.H., Hahn, K.M., Wang, G.G., Vedadi, M., Jin, J. An Orally Bioavailable Chemical Probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem. Biol. 8 (6):1324–1334, 2013.
 

MacNevin, C.J., Gremyachinskiy, D., Hsu, C.W., Li L., Rougie, M., Davis, T.T., Hahn, K.M. Environment-Sensing Merocyanine Dyes for Live Cell Imaging Applications. Bioconjug. Chem. 24(2):215-23, 2013.

Gulyani, A., Vitriol, E., Allen, R., Wu, J., Gremyachinskiy, D., Lewis, S., Dewar, B., Graves, L.M., Kay, B.K., Kuhlman, B., Elston T., and Hahn, K.M.  A biosensor generated via high-throughput screening quantifies cell edge Src dynamics. Nature Chem. Bio., 7: 437-444, 2011.

Shadpour, H., Zawistowski, J.S., Herman, A., Hahn, K.M., and Allbritton, N.L.  Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors. Analytica Chimica Acta, 696: 101-107, 2011. 

Abbas A, Roth BL. 2008. Protein engineering: electrifying cell receptors. Nature Nanotechnol 3:587-8

Alexander GM, Rogan SC, Abbas AI, Armbruster BN, Pei Y, et al. 2009. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63:27-39

Besnard J, Ruda GF, Setola V, Abecassis K, Rodriguiz RM, et al. 2012. Automated design of ligands to polypharmacological profiles. Nature 492:215-20

Carlsson J, Coleman RG, Setola V, Irwin JJ, Fan H, et al. 2011. Ligand discovery from a dopamine D3 receptor homology model and crystal structure. Nat Chem Biol 7:769-78

Conklin BR, Hsiao EC, Claeysen S, Dumuis A, Srinivasan S, et al. 2008. Engineering GPCR signaling pathways with RASSLs. Nat Methods 5:673-8

Dong S, Rogan SC, Roth BL. 2010. Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs. Nat Protoc 5:561-73

Fenalti G, Giguere PM, Katritch V, Huang XP, Thompson AA, et al. 2014. Molecular control of delta-opioid receptor signalling. Nature

Garner AR, Rowland DC, Hwang SY, Baumgaertel K, Roth BL, et al. 2012. Generation of a synthetic memory trace. Science 335:1513-6

Gray JA, Roth BL. 2002. Cell biology. A last GASP for GPCRs? Science 297:529-31

Huang HS, Allen JA, Mabb AM, King IF, Miriyala J, et al. 2012. Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature 481:185-9

Keiser MJ, Roth BL, Armbruster BN, Ernsberger P, Irwin JJ, Shoichet BK. 2007. Relating protein pharmacology by ligand chemistry. Nat Biotechnol 25:197-206

Keiser MJ, Setola V, Irwin JJ, Laggner C, Abbas AI, et al. 2009. Predicting new molecular targets for known drugs. Nature 462:175-81

Kokel D, Cheung CY, Mills R, Coutinho-Budd J, Huang L, et al. 2013. Photochemical activation of TRPA1 channels in neurons and animals. Nat Chem Biol

Kozikowski AP, Roth BL, Tropsha A. 2006. Why academic drug discovery makes sense. Science 313:1235-6

Laggner C, Kokel D, Setola V, Tolia A, Lin H, et al. 2011. Chemical informatics and target identification in a zebrafish phenotypic screen. Nat Chem Biol

Lin H, Sassano MF, Roth BL, Shoichet BK. 2013. A pharmacological organization of G protein-coupled receptors. Nat Methods 10:140-6

O'Connor KA, Roth BL. 2005. Finding new tricks for old drugs: an efficient route for public-sector drug discovery. Nat Rev Drug Discov 4:1005-14

Oury F, Sumara G, Sumara O, Ferron M, Chang H, et al. 2011. Endocrine regulation of male fertility by the skeleton. Cell 144:796-809

Roth B.L. 2000. Neuronal signal transduction: wasteland or the promised land? Science-STKE

Roth BL. 2007. Drugs and valvular heart disease. N Engl J Med 356:6-9

Roth BL, Marshall FH. 2012. NOBEL 2012 Chemistry: Studies of a ubiquitous receptor family. Nature 492:57

Roth BL, Sheffler DJ, Kroeze WK. 2004. Magic shotguns versus magic bullets: selectively non-selective drugs for mood disorders and schizophrenia. Nat Rev Drug Discov 3:353-9

Thompson AA, Liu W, Chun E, Katritch V, Wu H, et al. 2012. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485:395-9

Vardy E, Roth BL. 2013. Conformational ensembles in GPCR activation. Cell 152:385-6

Vedadi M, Barsyte-Lovejoy D, Liu F, Rival-Gervier S, Allali-Hassani A, et al. 2011. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat Chem Biol 7:566-74

Wacker D, Wang C, Katritch V, Han GW, Huang XP, et al. 2013. Structural features for functional selectivity at serotonin receptors. Science 340:615-9

Wang C, Jiang Y, Ma J, Wu H, Wacker D, et al. 2013. Structural basis for molecular recognition at serotonin receptors. Science 340:610-4

Wang C, Wu H, Katritch V, Han GW, Huang XP, et al. 2013. Structure of the human smoothened receptor bound to an antitumour agent. Nature

Wu H, Wacker D, Mileni M, Katritch V, Han GW, et al. 2012. Structure of the human kappa-opioid receptor in complex with JDTic. Nature