Section 9-1 National Tsing Hua University

CHEM5980

Signal transduction
• Communication is essential for the animals on the planet to live and interact with one
another. Likewise organism are comprised of cells and these cells need communicate
exert functions in an organism.
• Signal transduction at cell level relies on chemical signals. Reception a signal allows cell to
respond to the environment accordingly. Likewise sending out a signal is as important as
receiving signals.
• Signal transduction
is usually illustrated
in the form of a
diagram. Arrows
can mean activation
or interactions and
do not always imply
direct interaction.
• Lines that end with
short perpendicular
bar indicates
inhibition.
 Section 9-1 National Tsing Hua University
CHEM5980

Signal transduction
• Signal transduction may or may not result in gene transcription and further
translation into protein. The signals that transcribe gene are different from those
do not.
• The signal that lead to response to biochemical machinery existing in cell do not
require transcription process. As a result, such signal leads to rapid response. In
contrast, the signal lead to transcription would require entering nucleus and also
protein synthesis, which would take much longer to respond.
• Some signal involves both fast and slow responses.
 Section 9-1 National Tsing Hua University
CHEM5980

Cell contraction
• One of the fast signal transduction responses is muscle contraction.
• The movement of muscle is driven by the crawling of myosin filaments along
actin filaments in response to Ca2+ ions. Myosin protein groups into bundles with
the head group protrude from the bundles. The head groups are equipped with
ATP driven motor that crawls with each stroke driven by one ATP molecule.
• Each myosin motor is controlled by the state of phosphorylation, where
phosphorylated is on and dephosphorylated is off. Ca2+ binds and changes
conformation of Ca2+ sensing protein calmodulin, which then bind and activate
kinase for phosphorylation.
 Section 9-1 National Tsing Hua University
CHEM5980

Vesicle fusion
• Neural signal transmission require the release and receive of neurotransmitters. These
small molecules such as acetylcholine and dopamine are packed in vesicles so it can
release rapidly upon triggered by Ca2+ ion.
• A complex of protein is
responsible for holding the
vesicle close to membrane
but far enough not to
accidentally trigger the
release neurotransmitter.
• Upon Ca2+ binding to
synaptotagmin, the vesicle
is pulled to the membrane
and releases
neurotransmitters.
 Section 9-1 National Tsing Hua University
CHEM5980

Vesicle fusion
A general mechanism of membrane fusion by the interaction of soluble N-ethylmaleimide-
sensitive factor (NSF)-attachment protein receptors (SNARE) and Sec1/Munc18-like (SM)
proteins

Nat Med 2013, 19, 1227

 Section 9-1 National Tsing Hua University
CHEM5980

Cell signaling pathways

• Cell signaling can occur with in or between cells.
• By identifying how the signaling molecule travel for cell signaling, several types of
signaling such as autocrine, paracrine, intracrine and endocrine are defined.
• Autocrine signaling is that a cell secretes a chemical messenger that binds to
autocrine receptors on that same cell.
• Paracrine means a cell
produces a signal to induce
changes in nearby cells.
• Intracrine is the signaling
produces and receives within
a cell.
• Endocrine refers to that
secrete hormones directly
secreted into the circulatory
system to be carried towards
a distant target cells
J Cereb Blood Flow Metab 2000 20, 1393
 Section 9-2 National Tsing Hua University
CHEM5980

Signaling pathways involve transcription

• There may be thousands of signaling pathways that involve transcription in
human cells, however, these pathways can be categorized into only seven types.
• The seven types of pathways included nuclear receptors and other multistep
pathways including: two component pathway, receptor tyrosine kinases, trimeric
death receptors, G protein-coupled receptors, ion-channel receptors and gas
receptors.

, tastes, photon
 Section 9-2 National Tsing Hua University
CHEM5980

Methods to study genetics

• The study of genetics relied on
the identification of gene
(genotype) and the resulted
function (phenotype).
• Two broad direction was
exploited to study genetics. In
forward genetics randomly
introduce mutations into target
organism such as Drosophila.
The mutant phenotypes are
identified and then DNA is
sequenced to reveal the
genotype.
• In reverse genetics, specific
proteins are examined and the
mutations are introduced to
inspect their functions.

Nat Rev Genetics 2000, 1, 116

 Section 9-2 National Tsing Hua University
CHEM5980

Chemical genetics
• Small molecules provide another strategy to
understand the protein functions and the upstream
gene. For example, Rapamycin complex with FKBP12
then bind to mTOR. This interference suppresses
immune response, therefore function of mTOR is
understood.
• In the case of focal adhesion kinase (FAK), removal of
this protein is lethal so that animal lacking FAK cannot
be engineered. Selective FAK inhibitors can
temporally shut down the FAK to observe the
difference and predict its functions.

Nat Rev Genetics 2000, 1, 116

 Section 9-2 National Tsing Hua University
CHEM5980

Chemical genetics
• The chemical genetics is powerful strategy to investigate the function of specific genes
and proteins. However this method heavily relies on the availability of small molecules
specific for target protein.
• When a natural product
partner molecule is not
specific or potent enough
for the target protein,
synthetic small molecules
have to be used as a tools
for such investigation.
• As many of such proteins
are new and lacking the
information how small
molecule partner can be
designed, screen existing
compounds becomes a
common strategy to find
the right small molecule.
 Section 9-2 National Tsing Hua University
CHEM5980

Sphingosine 1-phosphate receptor

When patients have organ transplantation,
rejection is one critical issue to take care of.
Traditional immunosuppression treatment shut
down the lymphocyte activity and result in
impaired immune system. An alternative strategy
is sequester lymphocytes in lymph node to avoid
rejection but do not shut it down entirely.

http://www.neurosurgical.com/neuro_medical_info/medicines/

Science 2002, 296, 348

 Section 9-2 National Tsing Hua University
CHEM5980

Sphingosine 1-phosphate receptor antagonist

Nat Immunol 2005, 12, 1228

Nat Chem Biol 2006, 2, 434

 Sphingosine 1-Phosphate Receptor-1
(S1P1) Antagonists
• Sphingosine is named after Greek
mythological creature Sphinx in
1884 for its enigmatic nature due to
its enigmatic nature in controlling
many biological functions. Sphingosine
• Typical functions controlled by S1P
are listed below:
S1P phosphatase Sphingosine kinase
Regulate cell growth
Suppress programmed cell death
Regulate calcium concentration Sphingosine
1-phosphate
Heart / Vascular development
Vascular integrity S1P lyase
Lymphocyte migration

• The lymphocyte migration is most degradation

interesting function because it allows
tuning the extent of immune response.
 The functions of S1P receptors

S1P
S1P receptor type-1 (S1P1) is
responsible for the lymphocyte
migration function

Lymphocyte Cardiac Heart rate

migration development Pulmonary
Angiogenesis (Zebra Fish) epithelial
Pulmonary Neuronal integrity
endothelial dysfunction
integrity (mice) Science 2002, 296, 346
J Biol Chem 2004,
2005, 279,
280, 13839
9833
 S1P / agonists
The functionsand theirreceptors
of S1P specificity

S1P

S1P agonist
Immunosuppressing agent went clinical
FTY720P trial for kidney transplantation and
multiple sclerosis
S1P1 selective agonist,
SEW2871
immunosuppressant
EC50 (nM)

VPC23031
S1P 1.4 0.31 0.17 95 0.61

FTY720P 0.21 >10000 4.9 4.3 1.0

SEW2871 13 >10000 >10000 >10000 >10000

Science 2002, 296, 346

J Biol Chem 2004,
2005, 279,
280, 13839
9833
 Head group modifications

VPC23031
• VPC23031 is selective S1P1 antagonist but inactive in vivo possibly due to hydrolysis of
phosphate group.
• W123 was non-hydrolysable analog that designed based on the charge location.

SEW2871
alone
SEW2871
+ W123

Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH,
Parker I, Cahalan MD. Nat Immunol 2005, 6, 1228
Rosen group
 Experiment settings

2-photon
microscopy to
observe T cell
Thymus Marrow dynamics

Blood

Thoracic duct Tissue

Lymph
node

Explant mouse lymph node

Curr Opin Chem Biol 2003, 7, 461
Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH,
Parker I, Cahalan MD. Nat Immunol 2005, 6, 1228
 The lymphatic effects of agonist SEW2871 ex vivo

Velocity and transendothelial migration of T cells are inhibited by SEW2781,

but rapidly recovered following drug washout
In presence of SEW2871, T cell move laterally along sinus boundary or
away from the boundary
T cells cross between medullary cord and sinus more frequently at certain sites
Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH,
Parker I, Cahalan MD. Nat Immunol 2005, 6, 1228
Rosen, Cahalan & Parker group
 T cell numbers in medullary sinus and cord in explant lymph node

Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH,
Rosen, Cahalan & Parker group Parker I, Cahalan MD. Nat Immunol 2005, 6, 1228
 The reversal of agonist SEW2871 action by antagonist W123 ex vivo

W123 Restore T cells

velocity in medulla
that is treated with
agonist SEW2871

Antagonist W123 restores T-cell transendothelial migration

W123 restores T-cell velocity in medulla
W123 has no effect when applied alone

Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH,
Rosen, Cahalan & Parker group Parker I, Cahalan MD. Nat Immunol 2005, 6, 1228
Stromal gating model

Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH,
Parker I, Cahalan MD. Nat Immunol 2005, 6, 1228
 Second generation S1P1 antagonist
KD(nM)
45 Although W123 is a successful
ex vivo antagonist, effective
concentration can not be
690 maintained in vivo

Phosphate may enhances

W148 281
activity

W146 (R) 77 Only R form (W146) active,

W140 can be used as control
W140 (S) 4630 for in vivo studies

S1P + 10µM W146 W146 W146 showed similar activity

S1P + 10µM W140 W140
S1P alone as VPC23019

W146 maintain 270nM in

plasma at 5hr after 10mg/kg
injection

Sanna, Don Sanna MG†, Wang SK†, Gonzalez-Cabrera PJ, Don A, Marsolais D, Matheu MP, Wei SH, Parker I, Jo E,
Rosen group Cheng WC, Cahalan MD, Wong CH, Rosen H. Nat Chem Biol. 2006, 2, 434
 In vivo T cell migration under agonist SEW2871 and antagonist W146

Medulla Medulla
SEW SEW SEW + W146

Matheu, wei Sanna MG†, Wang SK†, Gonzalez-Cabrera PJ, Don A, Marsolais D, Matheu MP, Wei SH, Parker I, Jo E,
Cheng WC, Cahalan MD, Wong CH, Rosen H. Nat Chem Biol. 2006, 2, 434
Cahalan & Parker group
 W146 reverses agonist effect on blood lymphocyte number

W146 reverse S1P1 agonist effect on lymphopenia in vivo, further

support the proposed stromal gating model

Physiological concentration of S1P do not measurably alter the

constitutive lymphocyte recirculation

Sanna, Don Sanna MG†, Wang SK†, Gonzalez-Cabrera PJ, Don A, Marsolais D, Matheu MP, Wei SH, Parker I, Jo E,
Rosen group Cheng WC, Cahalan MD, Wong CH, Rosen H. Nat Chem Biol. 2006, 2, 434
 Antagonist W146 reverse agonist protection from VEGF capillary leakage

1. Evans blue dye injection (i.v.) to label plasma protein

2. Agonist / antagonist administered
3. Monitor VEGF induce capillary leakage

Gonzalez-Cabrera Sanna MG†, Wang SK†, Gonzalez-Cabrera PJ, Don A, Marsolais D, Matheu MP, Wei SH, Parker I, Jo E,
Rosen group Cheng WC, Cahalan MD, Wong CH, Rosen H. Nat Chem Biol. 2006, 2, 434
 Blockade of S1P1 induces spontaneous capillary leakage in lung

Evan’s blue dye

labeled plasma
protein

No agonist added, therefore W146 is competing basal S1P signal

Marsolais Sanna MG†, Wang SK†, Gonzalez-Cabrera PJ, Don A, Marsolais D, Matheu MP, Wei SH, Parker I, Jo E,
Rosen group Cheng WC, Cahalan MD, Wong CH, Rosen H. Nat Chem Biol. 2006, 2, 434
 Model for vascular integrity

W146 reverse the protection from VEGF-induced leakage by S1P1

selective agonist
W146 enhances basal capillary leak in a dose-dependent manner
S1P1 blockade by W146 induces spontaneous pulmonary edema

W146

Sanna MG†, Wang SK†, Gonzalez-Cabrera PJ, Don A, Marsolais D, Matheu MP, Wei SH, Parker I, Jo E,
Cheng WC, Cahalan MD, Wong CH, Rosen H. Nat Chem Biol. 2006, 2, 434
 Section 9-3 National Tsing Hua University
CHEM5980

Nuclear receptors
• Human transcription factor receptors are commonly refer to nuclear receptors,
which allow transcription upon receiving signals.
• Human genome encodes for 28 types of nuclear receptors. Steroid hormones are
well-known ligands for these receptors with high specificity and high affinity.
• Only 8 of the 28 types of receptors have their ligands found, the rest types are
orphan receptors, whose ligands are yet to be identify.
• One interesting receptor is liver
X receptor (LXR), which is
activated by two very different
ligands sterols and
monosaccharides.
 Section 9-3 National Tsing Hua University
CHEM5980

Nuclear receptors
• All nuclear receptors are intracellular so that all ligands for such receptors must be
either intracellular or cell permeable. Notably some nuclear receptors translocate
into nucleus.
• Estrogen receptor-α is one example that upon binding to the ligand estradiol, forms
dimer and complex with heat shock protein 90 (Hsp90). The dissociation of
estrogen receptor dimer from Hsp90 allows the active transport of the receptor
into nucleus.
• The estrogen receptor
carries nucleus localization
signal, which a short
peptide with specific
sequences. This peptide
binds to karyopherin β and
import to nucleus through
nuclear pore complex.
• Inside nucleus, ligand-
bound receptor binds to a
specific DNA sequence and
enhance transcription of
gonadotropin-releasing
hormone (GnRH).
 Section 9-3 National Tsing Hua University
CHEM5980

Nuclear receptors
• Another variant of nuclear receptor involves receptor heterodimerization, which
alters gene transcription.
• Trans-retinoic acid, a drug for cystic acne treatment, is a well-known example for
this type of receptors. This drug effects on cell growth especially on fetus and
therefore considered as potent teratogen. Its usage has been strictly controlled.
• Retinoic acid diffuses across
cell membrane and nuclear
membrane to reach nucleus
and binds to retinoic acid
receptor α (RAR α).
• The ligand-bound RAR α
further complex with
retinoid X receptor (RXR) as
heterodimer to bind to
specific sequence of DNA to
enhance and repress the
transcription of certain
genes.
 Section 9-3 National Tsing Hua University
CHEM5980

Nuclear receptors
• The DNA recognition of nuclear receptor is enabled by the Zn finger domain, which
only recognize 6-bp sequences AGAACA or AGGTCA. By the formation of receptor
heterodimer, the recognition expanded to two 6-bp sequences.
• Some of the heterodimeric receptors are in
head-to-head complex so that the sequence is
inverted.
• The length of base pairs between two 6-bp
sequences affect the relative location of the
recognition sites on B-form DNA duplex.
 Section 9-3 National Tsing Hua University
CHEM5980

Drosophila nuclear receptors

• The Structure of nuclear receptor are similar across different organisms. However
the ligand binds to these receptors may be different.
• In Drosophila the maturation from juvenile to adult is controlled by 20-
hydroxyecdysone and juvenile hormone III to ecdysone and ultraspiracle receptors
respectively.
• Plant also produce chemicals that mimic insect ligands to target insects signal
transductions. Fir tree produces terpene juvabione to interfere insect molting.
Likewise, synthetic pesticide tebufenozide mimics ecdysone to induce premature
molt.
 Section 9-3 National Tsing Hua University
CHEM5980

Engineering nuclear receptors

• Although these insect ligands and receptors are absent in human cells, ecdysone and
ultraspiracle receptors are homologs to human farnesoid X and retinoid X receptors
respectively. Moreover, ecdysone receptor forms heterodimer complex with human
retinoid X receptor.
• When human transfected with
ecdysone receptor plasmid and
treated with ligand, the ecdysone
receptor associate with human
retinoid X receptors and bind to DNA.
• When the ecdysone receptor
engineered to include mammalian
VP16 domain to recruit human
transcription machinery and point
mutations performed to recognize the
same DNA sequence as glucocorticoid
receptor, the engineered nuclear
receptor system respond to ecdysone
and turn on the transcription in
human cells.
 Section 9-3 National Tsing Hua University
CHEM5980

Steroids
• Given that a broad variety of cellular functions are controlled by steroid ligand on
cellular receptors and that steroids are very potent in inducing such effects, steroid
chemistry has become an important field of research.
• Current synthetic steroids use natural steroid isolated from soybean as starting
material.
• Besides the estrone conjugate isolated from pregnant mare urine, other steroids such
as glucocorticoids have been widely used in prescriptions.

Steroids from soybean

Steroids as therapeutics
 Section 9-3 National Tsing Hua University
CHEM5980

Nonsteroidal ligands for nuclear receptors

• Many natural ligands for nuclear receptors are nonsteridal, these ligands are also
widely used as drugs.
• One example pair is triiodothyronine and tetraiodothyronine, which are produced by
thyroid gland in the neck. Triiodothyronine controls metabolism throughout the body
by binding to nuclear receptors.
• The signaling from triiodothyronine increase the metabolic rate and the usage of
glucose and lipids. Moreover, physiological response such as heart rate, blood
pressure and body temperature are controlled by these ligands.
• To biosynthesis these ligands, iodine is required. If the iodine is deficient from diet,
thyroid gland proliferates and leads to goiter. Therefore KI has been added to grocery
salt.
• Iodine has strong affinity to thyroid gland, therefore in the case of nuclear accident, in
which radioactive 131I is released to environment, natural 127I are usually used to
prevent the uptake of deleterious 131I.
 Section 9-3 National Tsing Hua University
CHEM5980

Drugs targeting mutant nuclear receptors

• Vitamin D3, which is biosynthesized through photochemistry from 7-dehydro-
cholesterol, is essential ligand for vitamin D receptor that involved in proper bone
growth. Deficiency in vitamin D causes rickets.

• Mutation at the ligand-binding site of

vitamin D receptor are associated with
vitamin D-resistant rickets. The R274L
mutation removes one hydrogen bond
and cause 1000 time less in vitamin D
response.
• Modification with a benzyl group to 1-
hydroxyl group of vitamin D that fills
the void in the mutant restores 80% of
the inducible activity.