Structure, Function and Drug Discovery of the G-protein
Coupled Receptor Superfamily
Raymond Stevens1,2
The Scripps Research Institute1, La Jolla, CA 92037, USA
Shanghai Institute of Materia Medica2, Chinese Academy of Sciences
Shanghai 201203, China
G protein-coupled receptors comprise the largest family of
human proteins that communicate signals across the membrane
and recognize millions of diverse molecules such as adrenaline,
opioids, caffeine, dopamine, chemokines to name only a few.
Over the past 10 years we have built a process pipeline to
determine representative members of the G protein-coupled
receptor phylogentic tree in order to understand the similarities
and differences within this protein superfamily. In 2007, we
solved the crystal structures of the human β2-adrenergic receptor
bound to the partial inverse agonist carazolol and timolol at 2.4 Å and 2.8 Å resolution
and are now conducting NMR and HDX studies to understand receptor dynamics. In 2008,
we determined the structure of the human adenosine A2A receptor bound to the
antagonist ZM241385 at 2.6 Å resolution. More recently, we have determined the
structures of the human CXCR4 chemokine, human dopamine D3, histamine H1 receptor
structures, and agonist structure of the human adenosine A2a receptor. As part of a
biotechnology start-up to use the technologies for specific drug discovery, Receptos has
determined the structure of the human S1P1 receptor and the company now has a Phase I
clinical trial underway to treat multiple sclerosis. The collective structures provide a
high-resolution view of a human G protein-coupled receptor bound to diffusible ligands.
Ligand-binding site accessibility is enabled by the extracellular loops which are held out
of the binding cavity by a set of disulfide bridges and unique structural motifs. An
exciting discovery is the role of cholesterol in receptor stability and potential function.
Future studies include the determination of representative members from the different
branches of the G protein-coupled receptor phylogenetic tree including class A, B, and C
G protein-coupled receptors, as well as the receptors bound to agonists and G-proteins in
an activated state.
Raymond Stevens Professor, the Scripps Research Institute, USA; B.A., 1986, University
of Southern Maine; Ph.D., 1988, University of Southern California; NIH Postdoctoral
Fellowship, 1989-1992, Harvard University (advisor: William N. Lipscomb); Professor of
Chemistry, University of California, Berkeley, 1992-2000; Structural neurobiology and
G-protein coupled receptors; Tel: 858-784-9416, Fax: 858-784-9483, E-mail:
stevens@scripps.edu
Molecular Imaging Probes with Tunable Chemical Switches
Kazuya Kikuchi1,2
Graduate School of Engineering,1 Immunology Frontier Research Center,2 Osaka
University, Japan
One of the great challenges in the post-genome era is to clarify
the biological significance of intracellular molecules directly in
living cells. If we can visualize a molecule in action, it is
possible to acquire biological information, which is unavailable
if we deal with cell homogenates. One possible approach is to
design and synthesize chemical probes that can convert
biological information to chemical output.
Protein fluorescent labeling provides an attractive approach to study the localization
and function of proteins in living cells. Recently, a specific pair of a protein tag and
its ligand has been utilized to visualize a protein of interest (POI). In this method, a
POI is fused with a protein tag and the tag is labeled with the ligand connected to a
fluorescent molecule. The advantage of this protein labeling system is that a variety
of fluorescent molecules are potentially available as labeling reagents, and that the
protein tag is conditionally labeled with its fluorescent ligand. However, in the
existing labeling systems, there are some problems with the size of a protein tag, the
specificity of the labeling or fluorogenicity of labeling reagents. Protein tags for
labeling proteins of interest (POIs) with small molecule based probes have become
important technique as practical alternatives to the fluorescent proteins (FPs) for live
cell imaging. We have designed a protein labeling system that allows fluorophores
to be linked to POI. The protein tag (BL-tag) is a mutant class A -lactamase
(TEM-1) modified to be covalently bound to the designed specific labeling probes and
the labeling probes is consisted with a -lactam ring (ampicillin, cephalosporin)
attached to various fluorophores. A fluorogenetic labeling system can be designed
using the unique property of cephalosporin, which release leaving group by
subsequent reaction after opening the lactam ring. For further sophisticated
application, multicolor imaging was done by adopting the colorful fluorophores.
Kazuya Kikuchi Professor, Graduate School of Engineering and Immunology and
Frontier Research Center (double appointments), Osaka University; B.S. 1988,
University of Tokyo; Ph.D. 1994, University of Tokyo (advisor: Masaaki Hirobe);
Postdoctoral Training, 1994-1995, University of California, San Diego (advisor:
Roger Y. Tsien) 1995-1996, the Scripps Research Institute (advisor: Donald Hilvert);
Chemical biology; Design, synthesis and biological application of molecular imaging
probes;
Tel:
81-6-6879-7924,
Fax:
81-6-6879-7875,
E-mail:
kkikuchi@mls.osaka-u.ac.jp
Perturbing Angiogenesis Using Small Molecules
Ho Jeong Kwon
Chemical Genomics National Research Laboratory, Department of Biotechnology,
Translational Research Center for Protein Function Control, College of Life Science
and Biotechnology, Yonsei University, Seoul 120-752, Korea
Small molecules have been successfully utilized as molecular
probes to decipher molecular and cellular functions of their
binding proteins in a given biological phenotype of interest.
This ligand and receptor information further facilitates
structure based better drug design and development. My
laboratory has developed and applied this powerful potential
of small molecules to explore the complicated and
multi-components involved biological system such as
angiogenesis. From our continuing efforts to discover new
small molecules perturbing angiogenesis, a number of structurally distinct small
molecules such as terpestacin, curcumin, HBC, HNHA, OMe-Syn, and NHOBTD
have been identified from our synthetic and natural products library on the basis of
their phenotype suppressing activity toward the endothelial cells. Direct binding
proteins of these small molecules were identified using phage display biopanning
consisting of human whole cDNA expressing T7 phage library. As the results,
UQCRB, aminopeptidase N, calmodulin, and histone deacetylase were discovered as
the target proteins of these small molecules. New insights from these small molecules
and target proteins in respect to angiogenesis have enabled us to uncover new
molecular and signaling mechanisms underlying angiogenesis and to translate into the
development of new therapeutics towards angiogenesis related diseases. In this
presentation, our activities on systemic investigations toward angiogenesis using
small molecules will be provided by introducing our terpestacin and its target protein,
UQCRB, as one of case studies.
Ho Jeong Kwon Professor, Department of Biotechnology, Yonsei University; B.S.
1984, Seoul National University; Ph.D. 1995, Tokyo University (advisor: Teruhiko
Beppu and Sueharu Horinouchi); Postdoctoral Training, 1995-1998, Harvard
University. (advisor: Stuart L. Schreiber); Chemical biology, genomics, proteomics;
Discovery of bioactive small molecules and their targets, and application towards
understanding of biological systems; Tel: 82-2-2123-5883, Fax: 82-2-362-7265,
E-mail: kwonhj@yonsei.ac.kr
Make Invisible Visible: a Diversity Oriented Fluorescence Library
Approach (DOFLA)
Young-Tae Chang
Department of Chemistry, National University of Singapore, Singapore
With the successful result of Human Genome Project, we are
facing the problem of handling numerous target genes whose
functions remain to be studied. In chemical genetics, instead
of using gene knock-out or overexpression as in conventional
genetics, a small molecule library is used to disclose a novel
phenotype, eventually for the study of gene function. The
currently popular affinity matrix technique is challenging
because the transformation of the lead compound into an
efficient affinity molecule without losing the biological activity is not easy, requiring
intensive SAR studies. To surrogate the well known problem, our group has
developed a linker tagged library and has successfully identified multiple target
proteins so far. While successful, the affinity matrix technique requires a breakdown
of the biological system to pool the proteins into one extract, which inherently
introduce a lot of artifacts.
As the next generation of tagged library, we are currently developing fluorescence
tagged libraries for in situ target identification and a visualization of the biological
events using Diversity Oriented Fluorescence Library Approach (DOFLA). The
basic hypothesis is DOFLA of the same fluorescence scaffold, but with various
diversity elements directly attached around the core, may selectively respond to a
broader range of target proteins in intact biological system and facilitate the
mechanism elucidation and target identification. The high throughput strategy using
colorful chemical genetics for stem cell study will be discussed.
Young-Tae Chang was born in Busan, Korea, in 1968. He studied chemistry in
Pohang University of Science and Technology and received his B.S. in 1991. After
one and half years of army service in Korea, he started his graduate study at
POSTECH and received a Ph.D. in 1997 under the supervision of Prof. Sung-Kee
Chung, working on the divergent synthesis of all possible regioisomers of
myo-inositol phosphates. He did his postdoctoral work with Prof. Peter Schultz at UC
Berkeley and The Scripps Research Institute. In 2000, he was appointed assistant
professor at New York University and promoted to associated professor in 2005. He
received the NSF Career award in 2005 and his research interests have been chemical
genetics, molecular evolution, and artificial tongues. In September, 2007, he moved
to National University of Singapore and Singapore Bioimaging Consortium. He is
running Medicinal Chemistry Program of NUS as the leader, and Lab of bioimaging
Probe Development at SBIC, Biopolis. He published more than 180 scientific
papers/3 books and filed 30 patents so far; Tel: 65-6516-6774, E-mail:
chmcyt@nus.edu.sg
Synthesis and Immunology Evaluation of MUC1 Glycopeptide
Vaccine
Yan-Mei Li
Department of Chemistry, Key Lab of Bioorganic Phosphorus Chemistry & Chemical
Biology (Ministry of Education), Tsinghua University, Beijing 100084, China
The tumor associated mucin MUC1 is over-expressed in
most epithelial tumor tissue. The extracellular part of MUC1
contains a large number of tandem repeat sequence of
HGVTSAPDTRPAPGSTAPPA, which includes five
potential O-glycosylation sites. On tumor cells, the
glycosylation pattern of MUC1 is distinctly changed
compared with normal cells, which makes MUC1 an
attractive target for cancer immunotherapy. Typically, the
O-glycosylation on the tumor cells is truncated, resulting in
the Thomsen-Friedenreich antigen (T antigen), its precursor
(Tn antigen), and their sialylated forms. Together with these tumor-associated
carbohydrate antigens, the backbone peptide epitopes of MUC1 are also exposed.
However, as B-cell epitopes, the immunogenicity of these glycopeptides is low. To
induce sufficiently strong immune response, the synthetic glycopeptides need to be
conjugated with an immunostimulating component.
Herein, the MUC1 glycopeptides bearing Tn, T, STn and 2,6-ST antigen at the
sites of T9 and S15 were synthesized by microwave assisted solid-phase peptide
synthesis strategy. After deprotection of the carbohydrate portion, these synthetic
glycopeptides were conjugated to the carrier protein bovine serum albumin (BSA) via
a triethylene glycol spacer. These synthetic vaccines were immunized with balb/c
mice. Strong immune responses were induced by these vaccines. Except the IgM
antibody, strong IgG type titers were also observed. The mouse sera reacted strongly
with native MUC1 expressed on MCF7 human breast cancer cell line, which was
examined by flow cytometry.
Yan-Mei Li Professor, Director of Key Laboratory of Bioorganic Phosphorus
Chemistry & Chemical Biology (Ministry of Education). She graduated from
Tsinghua University and received Ph.D. in 1992 under the supervision of Professor
Yu-Fen Zhao. Then she worked with Professor H. Waldmann as a post-doctoral fellow
in Institute of Organic Chemistry, University of Karlsruhe, Germany. She was
promoted to Full Professor in 1998. Her research interests focus on synthesis of
peptide conjugates and effects of post-translational modifications on the structure and
functions of peptides and proteins; Tel: 86-10-62796197, E-mail:
liym@mail.tsinghua.edu.cn
Chemical Epigenetics
Minoru Yoshida
Chemical Genomics Research Group, RIKEN Advanced Science Institute, Japan
Chromatin modifications serve as the important epigenetic marks
responsible for inheritable changes in gene expression without
changes in the DNA sequence, which ensure the unique gene
expression patterns in differentiated tissues. The aberrant
chromatin modifications, which are caused by the
deregulation of factors that mediate the modification
installation, removal and/or interpretation, actively contribute
to human cancer. Indeed, histone deacetylase (HDAC) has
been shown to associate with a variety of oncogenic factors
that downregulate differentiation or tumor suppressor
functions at the specific genetic loci. HDAC inhibitors can
reverse the aberrant gene expression, and have been shown to have a therapeutic activity
in cancer treatment. During the course of our mode-of-action studies on anticancer
natural products, we have identified trichostatin A (TSA), trapoxin (TPX), and FK228
(Romidepsin) as the potent and specific HDAC inhibitors.
The long-term goal would be reversal or tuning of aberrant epigenetics occurring in
the cancer tissues by the combined use of the small molecules that modulate chromatin
modifications. To this end, we are currently making great effort to establish novel
systems for detecting and evaluating the activity of cellular factors that regulate writing,
reading and erasing of the epigenetic chromatin marks. These include in vivo imaging
probes for monitoring dynamic changes in histone acetylation in living cells, which
allows detection of not only HDAC inhibitory activity but also activity to interfere with
bromodomain binding to histones acetylated at specific sites. Furthermore, we
constructed in vitro fluorescent assays that enable high-throughput screening for histone
methyltransferases and demethylases.
Minoru Yoshida Group Director, Chemical Genomics Research Group, RIKEN
Advanced Science Institute; B.S. 1981, University of Tokyo; Ph.D. 1986, University of
Tokyo (advisor: Teruhiko Beppu); Assistant and Associate Professor, 1986-2002,
University of Tokyo; Chief Scientist, 2002-present, RIKEN; Chemical genetics;
Discovery and target identification of bioactive small molecules; Tel: 81-48-467-9516,
Fax: 81-48-462-4676, E-mail: yoshidam@riken.jp
Chemical and Systems Biology for the Discovery and Validation
of Druggable Targets
Sunghoon Kim
Medicinal Bioconvergence Research Center
Department of Molecular Medicine and Biopharmaceutical Sciences
Seoul National University, Seoul, Korea
Medicinal Bioconvergence Research Center (Biocon) was
established in 2010 under the program called global frontier
initiated by the Ministry of Education, Science and Technology
(MEST), Korea. This project was designed to innovate drug
discovery process by combining biology and chemistry with other
multiple disciplines for rapid discovery and validation of
therapeutic targets and drug leads so that they can be delivered to
industry. In Biocon, chemical biology is often employed to validate the efficacy of the
disease-associated genes and proteins that have been identified from integrated
approaches of genomics, proteomics and bioinformatics. In particular, chemicals are
screening to modulate protein-protein interactions or alternative splicing that show
therapeutic potential. Here we show a few case studies that proved useful to develop
novel chemicals that show a potential as novel anti-cancer therapeutics.
Sunghoon Kim Professor, Seoul National University; Director, Medicinal
Bioconvergence Research Center; B.S. 1981, Seoul National University; M.S. 1983,
Korea Advanced Institute of Science and Technololgy; Ph.D. 1991, Brown University;
Postdoctoral Training 1991-1994, MIT; Molecular and cell biology for target
discovery; Tel: 82-2-880-8180, Fax: 82-2-875-2621, E-mail: sungkim@snu.ac.kr
Small Molecule Tools for Cell Biology and Cell Therapy
Motonari Uesugi
Institute for Integrated Cell-Material Sciences, Kyoto University, Japan
In human history, bioactive small molecules have had
three primary uses: as medicines, agrochemicals, and
research tools. Among them, what our laboratory has done
in the past was the discovery and use of research tools for
biological investigations. In addition to tool discovery, our
laboratory has recently become interested in exploring
another application of small molecules: small molecule
tools for cell therapy. Although small molecule drugs will
continue to be important, cell therapy will be a powerful
approach to curing difficult diseases that small molecule
drugs are unable to handle. However, there are a number
of potential problems in bringing cell therapy technologies to the clinic, including
high cost, potential contamination, low stability, and tumorigenesis. Stable,
completely defined small molecule tools, which are usually amenable to cost-effective
mass production, may be able to help the clinical use of cell therapy.
Through screening chemical libraries, we have been discovering unique synthetic
molecules that modulate or detect fundamental characteristics of human cells useful
for cell therapy. Some of such molecules may serve as tools for cell engineering or
cell therapy as well as basic cell biological research. This presentation provides a
quick overview of our recent research programs with a special emphasis on the
discovery and utilization of two small molecules: one for basic cell biology research,
and the other for cell therapy applications.
Motonari Uesugi Professor, Institute for Integrated Cell-Material Sciences, Kyoto
University; B.S. 1990, Kyoto University; Ph.D. 1995, Kyoto University. (advisor:
Yukio Sugiura); Postdoctoral Training, 1995-1998, Harvard University. (advisor:
Gregory L. Verdine); Chemical biology; Discovery and design of bioactive small
molecules, and their usage for biological investigations; Tel: 81-774-38-3225, Fax:
81-774-38-3226, E-mail: uesugi@scl.kyoto-u.ac.jp
Molecular Diversity and Bioprobes for Chemical Biology
Seung Bum Park
Department of Chemistry and Department of Biophysics & Chemical Biology
Seoul National University, Seoul, 151-747, Korea
The importance of molecular diversity has been clearly
recognized to identify specific bioactive small molecules
for the elucidation of mysterious biological processes.
The concept of diversity-oriented synthesis (DOS) was
introduced for efficient population of molecular diversity
in untapped chemical space using complexity-generating
synthetic route. To address these unmet needs for
maximizing molecular diversity with high relevance in
biological
space,
we
pursued
privileged-substructure-based DOS (pDOS) strategy to
emphasize the importance of maximized skeletal
diversity through the creative reconstruction of core skeletons containing privileged
substructures for the construction of a drug-like polyheterocycle library. Our divergent
pDOS strategy can provide an efficient approach for the discovery of novel
small-molecule modulators with excellent specificity in chemical biology and drug
discovery.
Through screening chemical libraries, we have been discovering unique drug-like
molecules that modulate or detect fundamental characteristics of human cells useful
for cell therapy. Some of such molecules may serve as tools for cell engineering or
cell therapy as well as basic cell biological research. We also discovered the several
novel fluorescent bioprobes to monitor the specific biological events and subsequently
applied them for phenotype assay via image-based high-content screening. This
presentation provides a quick overview of our recent research programs with a special
emphasis of pDOS strategy and application of novel fluorescent bioprobes.
Seung Bum Park Associate Professor, Department of Chemistry and Department of
Biophysics & Chemical Biology, Seoul National University; B.S. 1993, Yonsei
University; M.S. 1997, Yonsei University; Ph.D. 2001, Texas A&M University
(advisor: Robert F. Standaert); Postdoctoral Training, 2001-2004, Harvard University
(advisor: Stuart L. Schreiber); Chemical biology, diversity-oriented synthesis,
fluorescent bioprobes, medicinal chemistry; Design and construction of drug-like
small molecule library, discovery of novel bioactive compounds and fluorescent
bioprobes, and their usage for biological investigations; Tel: 82-2-880-9090, Fax:
82-2-884-4025, E-mail: sbpark@snu.ac.kr
Imaging and Control of Biomolecules in Live Cells Using
Genetically Encoded Proteins
Takeaki Ozawa
Department of Chemistry, School of Science, The University of Tokyo, Japan
Photo
Fluorescent and bioluminescent proteins are now widely
used for detection of small molecules and various
intracellular events in living cells. Such analysis
depends on engineered protein-based probes with higher
sensitivity and selectivity. The probes can be entirely
genetically encoded and can comprise fusions of
different proteins or domains. The probes are quite
useful for screening new chemical compounds that
regulate intracellular signaling.
My laboratory has been developed various kinds of
genetically-encoded fluorescent and bioluminescent
probes for visualizing many intracellular events in living cells and animals. I herein
describe a novel design of engineered luminescent proteins such as GFP and
luciferases; the principle is based on reconstitution of the split-protein fragments
(reporters) when they are brought sufficiently close together. To demonstrate the
usefulness of the split reporters, I will show the reporters for imaging dynamics of
endogenous mRNA, protein release from mitochondria and protein-protein
interactions in living cells. We further developed another design of reporter proteins; a
cyclic luciferase by protein splicing to monitor protease activities in living mice. I will
describe possibilities of these probes to screen chemical compounds and highlight
some potential applications.
Takeaki Ozawa Professor, Department of Chemistry, School of Science, the
University of Tokyo; B.S. (1993), M.S. (1995) and Ph.D. (1998), the University of
Tokyo; Research Associate (1998-2002) and Lecturer (2002-2005), the University
of Tokyo; Associate Professor, the Institute for Molecular Science, Japan, 2005-2007;
Development of analytical methods for visualizing and controlling functions of
biomolecules in living subjects; Tel: 81-3-5841-4351, Fax: 81-3-5802-2989, E-mail:
ozawa@chem.s.u-tokyo.ac.jp
Cyclobutane Derivatives as Novel Non-peptidic Small Molecule
Agonists of Glucagon-like Peptide-1 Receptor
Ming-Wei Wang
The National Center for Drug Screening, Shanghai 201203, China
Type 2 diabetes is emerging as one of the largest health
issues worldwide with an estimated 23.6 million children
and adults (7.8% of the population) affected in the United
States alone. Glucagon-like peptide-1 (GLP-1) has
proven to be an efficacious agent to combat this serious
and life-long disease. A novel cyclobutane class of
non-peptidic GLP-1 receptor agonists, exemplified by
Boc5, was identified using receptor binding and cAMP
response element-driven reporter gene assays. The
structures of Boc5 and its three isomers were elucidated
by NMR, HRESIMS, and X-ray crystallography. A series
of structural modifications were also made based on the
core structure of Boc5 with different substitution groups at the west and east ends.
In vitro characterization of these analogues demonstrated that the cyclobutane core
and the two carboxylic groups were essential for the bioactivity and modifications
such as decreasing the size of the ring and conversion of the acids to amide and ester
led to the total loss of activity. The Boc and the 2-thiophenyl groups were
well-tolerated with compound 13 as the optimum that consistently displayed more
potent GLP-1 activities (than Boc5) both in vitro and in vivo. Preliminary
structure-activity relationship studies suggested that the cyclobutane analogues may
serve as a starting point for the development of new GLP-1 mimetics.
Ming-Wei Wang Professor, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences; M.B. 1982, Shanghai First Medical College; Ph.D. 1989, University of
Cambridge (advisor: Sir Brian Heap); Postdoctoral Training, 1989-1990, University
of Cambridge Clinical School (advisor: David White); Chemical biology; Drug
discovery and elucidation of mechanisms of action relative to bioactive small
molecules;
Tel:
88-21-50800598,
Fax:
86-21-50800721,
E-mail:
wangmw@mail.shcnc.ac.cn