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TRP ion channels in taste
perception
Angela Bassoli
DeFENS
Sezione di Scienze Chimiche e Biomolecolari
Associazione Genetica Italiana
Scuola di Genetica in Cortona – 13 e 14 Giugno 2017
The TR(I)P active compounds:
cooling, hot, tingling, numbing and
the other somatosensory gustative
sensations
A.Bassoli-Cortona 13/06/2017
from “unusual” tastes and gustative
sensations
inside human body and its functions
towards medicin, nutrition, neurology,
physiology and more..
TR(I)P as a journey :
A.Bassoli-Cortona 13/06/2017
HOT
COOLING
PUNGENT
LACRIMATING
TINGLING
NUMBING…
A new family of receptors: the
somatosensory (chemesthetic)
gustative sensations
TR(I)P
ion channels,
indirect
activation
organic
molecules
or
physical stimuli
as touch,
temperature
change...
TR(I)P : Transient Receptor (Ionic) Potential
A.Bassoli-Cortona 13/06/2017
Ca 2+ release
Activation
(conformational change?)
Stimulus
(physical or
chemical)
How does a TRP receptor work?
signal
A.Bassoli-Cortona 13/06/2017
How many TRP receptors do we have?
The phylogenetic tree of TRP receptors
D.E. Clapham, Nature, 426, 517-524, 2003
A.Bassoli-Cortona 13/06/2017
Where the TRP channels are?
TRPs are quite conserved over evolution
antennae nose
inner
ear
mouth
skin
vibrisse
TRPs are spreadly diffused in many cells/organs
A.Bassoli-Cortona 13/06/2017
What do the TRPs do?
•Perception of “unusual” and “strong” gustative
sensations (chemesthesis, somatosensory)
•Perception of external signals from environment and
from other organisms (sensing) : temperature, light,
sound, touch, movement, pheromones…
•Perception of pain stimuli
TRPs are crucial
sensors for self
defense
mechanisms! A.Bassoli-Cortona 13/06/2017
TPRs: a rapidly
moving topic in
the literature
1293
2010 A.Bassoli-Cortona 13/06/2017
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TRP Channels: From Sensory Signaling to
Human Disease
September 26-27, 2009
Karolinska Institutet, Stockholm
Speakers
Craig Montell,Johns Hopkins Univ. School of Medicine
Bernd Nilius, Katholieke Universiteit
David Clapham, Harvard Medical School
Md.Shahidul Islam Karolinska Institutet
Ardem Patapoutian, Scripps Research Institute
Andrea Fleig, University of Hawaii
Michael Tymianski,Toronto Western Research Institute
Barbara Ehrlich, Yale School of Medicine
David Julius, University of California, San Francisco
Michael Caterina, Johns Hopkins Univ. School of
Medicine
X.Z. Shawn Xu, University of Michigan
Indu S. Ambudkar, NIDCR, National Institutes of Health
René J.M. Bindels, Radboud Univ. Nijmegen Med.
Centre
Thomas Gudermann, Ludwig-Maximilians-Universitaet
Baruch Minke, Hebrew University of Jerusalem
Andreas Lückhoff,University Hospital, Aachen
Peter Zygmunt, Lund University
A.Bassoli-Cortona 13/06/2017
Taste and temperature: TRPVs, TRPM8, TRPA1
COOL HOT
T (°C)
Some TRP channels are involved in the detection of high and
low temperature. The activation can be either induced by
physical and chemical stimulation.
A.Bassoli-Cortona 13/06/2017
A famous TRP: the vanilloids receptor TRPV1
The Atzech were
treating nasal
congestion with
hot pepper
Capsicum described in
“New Kreuterbuch” by
Leonhart Fuchs (1543)
TRPV1 was
cloned in 1997 by
David Julius
A.Bassoli-Cortona 13/06/2017
1957: Nicholas Jancsò
hypothesised the existence of a
specific receptor
TRPV1
A.Bassoli-Cortona 13/06/2017
TRPV1 is a thermosensitive ion channel activated by noxious heat (>43 °C),
low pH (<5.4) and a wide range of chemical stimuli like capsaicin and its
analogs (resiniferatoxin), anandamide and other arachidonic acid
metabolites.
A.Bassoli-Cortona 13/06/2017
A molecular model of the C-terminus of
TRPV1 was generated using the restraint-
based homology modelling method. The C-
terminus part was built using the crystal
structure of fragile histidine triad protein as
a template. Sequence homology between the
target (TRPV1) and the template (FHIT) is
44% . Other templates have been used for
modelling different part of the protein.
Crystal structures of several ATP-binding
cassette (ABC) transporters were used for the
modelling of the nucleotide-binding site. The
Walker A motif was built using the human
cystic fibrosis transmembrane regulator
(CFTR), MalK, the transporter associated with
antigen processing (TAP1), the ATP-binding
subunit of histidine permease (HisP), and
MJ0796, a bacterial ABC transporter cassette.
Hanson et al, Biophys J., 2015, 108,1425-34
L. Grycova et al., Archives of
Biochemistry and Biophysics 465
(2007) 389–398
Several models have been proposed for the binding site of capsaicin and
other agonists as RTX and calmodulin.
A.Bassoli-Cortona 13/06/2017
Cao, Liao and Julius, Nature 2013, 504,113-18.
TRPV1 molecular modelling of the tetramer
The cooling ion channel: TRPM8
It is activated by cold
temperatures and by some
chemicals able to generate the
cooling sensation as menthol,
pulegone and eucaliptol.
TRPM8 owns to
the subfamily
of Melastatin
TRP receptors.
The most important studies have been done on menthol
and a very large number of its analogues prepared by
synthesis for the food industry but especially for
pharmaceutic and cosmetic applications.
A.Bassoli-Cortona 13/06/2017
OH
Mentha arvensis
(-) Menthol and some of
its synthetic derivatives
O
O
OH
HO
O OOH
O
OH
OH
HN
O
frescolat MGA frescolat MLisolpulegol
WS-3coolant agent 10
A.Bassoli-Cortona 13/06/2017
N
NH
NO2
OH
O
Other cooling compounds: ICILIN
A synthetic compound used to induce “shaking” in animals
for clinical studies. (E. T. Wei, J. Pharm. Pharmacol., 35, 110-112, 1983).
Not used for food applications. Cooling activity reported,
some doubts about its evaluation in sensory analysis.
Icilin activates TRPM8 in vitro but it shows an extremely
variable latency followed by extensive desensitization.
(Julius t al, Neuron, Vol. 43, 859–869, 2004).
(-)-mentol:
icilin: LD50 > 1500mg/Kg
LD50 = 700mg/Kg
(200x)
A.Bassoli-Cortona 13/06/2017
O
N
O
N
O
O
N
Cooling compounds from beer: the cyclic ketoenamines
These natural compounds were isolated from beer in 2001
and extensively studied. Chemically very unstable , data
not very reproducible. No in vitro data.
1. Wei E.T., Seid D.A., J. Pharm. Pharmacol., 1983, 35, 110-112
2. Ottinger H., Bareth A., Hofmann T., J. Agric. Food Chem., 2001, 49, 1336-1344
3. Ottinger H., Soldo T, Hofmann T., J. Agric. Food Chem., 2001, 49, 5383-5390
3-MPC (0.03X) 5-MPC (0.15X) DMPF (0.01X)
A.Bassoli-Cortona 13/06/2017
Other cooling compounds: cooling lactams
N
NH
NO2
OH
O
O
N
O
N
O
O
N
OH
N
O
OH
N
O
OH
N
NHO
OH
N
NH
O
icilina
3-MPC DMPF 5-MPC
Lead compounds
Some new synthetic derivatives have been obtained by
drug design approach:
A. Bassoli, G. Borgonovo, G. Busnelli, G. Morini , Synthesis of a new family of N-aryl
lactams active on chemesthesis and taste. Eur. J. Org. Chem., 2006, 1656- 1663).
Derivatives
No in vitro activation on TRPM8.
AVENANTHRAMIDES: “fresh” vs “cooling”.
HO
R1
O
NH COOH
R2
R1=H; R2=HR1=H; R2=OH
R1=OCH3; R2=HR1=OCH3; R2=OHR1=OH; R2=OH
A.Bassoli-Cortona 13/06/2017
Cooling compounds from AVENA SATIVA (oat)
SAR studies with 70
known cooling cmps
Avenanthramides constitute a well defined cluster together with icilin:
therefore we decided to develop an efficient synthetic pathway for
avenanthramides.
(-)-menthol
icilin
avenanthramides
cyclic a-ketoenamines
A.Bassoli-Cortona 13/06/2017
Four natural-like compounds and eight synthetic compounds were
obtained. Again, no activity on TRPM8 in in vitro assays.
R1
O
NH R
3
R2
R5
R4
R1
R2 R
3 R
4 R
5 ppm sensory descriptors
OHOHOHOH
OMeOMe
HHHHHH
HH
OMeOMe
HOMe
HH
OHOHOHH
COOHCOOHCOOHCOOHCOOHCOOHCOOHCOOHCOOH
HHH
HHHHHHHHH
COOHHH
HOHH
OHHHH
OHHH
COOHCOOH
25
26
27
28
29
30
31
32
33
34
35
36
21071202105232182236212232272184218
cooling, bittermasking ethanol tasteunpleasantsligthly coolingunpleasant, sligtlhy fruityslightly bittertastelessslightly cooling, astringentslightly bitterslightly bittertastelesstasteless
SENSORY EVALUATIONS
A. Bassoli, G. Borgonovo, G. Busnelli, G. Morini “Use of amide derivatives as taste-
modifying agents, flavouring compositions and products containing them” .
PTC/IB2005/000845 (12.04. 06)
TRPM8 : modelling
Fig. 2. Structure of the TRPM8 monomer
coloured by segments (colour code: N-T: blue;
S1: red; EL1: azure; S2: grey; CL1: brown; S3:
yellow; EL2: gold; S4: grey; CL2: green;
S5:white; EL3: pink; S6: azure; C-T: violet)
showing the solenoid shape of very large N-
terminal domain and the C-terminus which is
packed between N-terminus and the
transmembrane bundle composed by six
helices (S1–S6).
A. Pedretti, C. Marconi, I. Bettinelli, G. Vistoli
Biochimica Biophysica Acta, 2008 A.Bassoli-Cortona 13/06/2017
The TRPM8 gene (also called Trp-p8),
originally cloned as a prostate-specific
protein, encodes for a 1104-residue
transmembrane protein, whose functional
quaternary structure is a homotetramer
channel with modular structure and
allosteric gating.
Binding
site
Putative complex between menthol and
TRPM8.
The main interaction stabilizing the
complex
is the H-bond between Tyr745 and the
ligand's hydroxy group, while the apolar
residues are involved in hydrophobic
contacts.
Icilin is a quite symmetric molecule; it
can also assume an overturned pose in
which the nitrophenyl moiety replaces the
menthol scaffold contacting Tyr745 and
the phenol ring interacts with Asn799.
Both poses realize a quite identical
interaction pattern although docking
scores suggest that the binding mode
depicted in Fig. 4B is the preferred one.
TRPM8 is overexpressed in androgen dependent prostate
and bladder cancer cells. Therefore, compounds able to
act as agonists and antagonists can have important
applications in medicine.
A.Bassoli-Cortona 13/06/2017
TRPM8: not just beer and shave
for cool men’s health
TRPA1: a multitask receptor
TRPA1 is a single member of the TRPA
(ankyrin) subfamily.
Only one in mammals. Two members
(dTRPA1 and pyrexia) in Drosophila.
Large ankyrin domain (18 in TRPA1).
Expressed in nociceptors, Dorsal Root
Ganglia -DRG, trigeminal, inner ear and
hair cells.
Role in trasduction of mechanical stimuli;
Hypersensitivity to touch and heat/cold
induced by inflammation processes.
Specific role in pain perception A.Bassoli-Cortona 13/06/2017
ginger
mustard
Cruciferae
(broccoli,
cabbage,
brussel sprouts,
rocket..)
clove Perilla frutescens
(kaennip, shiso) onions
oregano
…and
many
more!
TRPA1 is a thermoreceptor, activated by noxious cold.
It is also activated by many
compounds that have chemesthetic
properties (pungent, irritant,
lacrimogen etc.)
A.B
asso
li-Co
rton
a 1
3/0
6/2
01
7
N SS
O
S S CHO
CHO
allylisothiocyanate
(mustard oil) allicin (garlic)
diallylidisulfide
(onions)
cinnamaldehyde
(cinnamon)
acrolein
(thermal byproduct)
C S
HN
O
R
R = H alpha-sanshool
R = OH alpha-hydroxy-sanshool
The structure of some known agonists of TRPA1
from food:
TRPA1 is also activated by icilin and THC; it is insensitive
to menthol and capsaicin.
A.Bassoli-Cortona 13/06/2017
Isothiocyanates are usually present in plants as glucosinolate, from
which they are relased by hydrolysis with mirosinase after disruption of
cell tissues:
RC
N
SHGlc
OSO3-
mirosinasiglucosio
RC
NOSO3
-
SH
aglicone
R N C S
isotiocianatiR
nitrile
R S
tiocianato
H2C
HCS
CH2n
cianoepitioalcani
C N C NC N
O
H
HO
H
HO
H
HOHH
S
OH
N
OSO2O-
R
a glucosinolate
A.Bassoli-Cortona 13/06/2017
P.Geppetti, D.Julius et al, PNAS, 2007
Pain therapy; allergy mechanisms
TRPA1, TRPV1 and pain
TRPA1 and TRPV1 are
both expressed in the
same nociceptor neurons.
Their agonists can block
the pain generated by
inflammatory processes
A.Bassoli-Cortona 13/06/2017
Mihara S. et al., The role of flavor and fragrance chemicals in TRPA1 (transient receptor
potential cation channel, member A1) activity associated with allergies. Allergy, Asthma, and
Clinical Immunology 2015;11(1):11.
Paul A. S. Breslin et al. J.Neuroscience, 2011
In vitro oleocanthal selectively activates the hTRPA1 channel in HEK 293
cells; its ability to excite the trigeminal nervous system in rodents requires
a functional TRPA1. Ibuprofen, an analgesic which elicits the same
restricted pharyngeal irritation as oleocanthal, also specifically excites
rodent sensory neurons via TRPA1.
A.Bassoli-Cortona 13/06/2017
TRPA1 and food: olive oil
oleocanthal ibuprofen
The aromatic food plant P. frutescens is rich in very
active TRPA1 agonists:
O
O
OH
Perilla frutescens, kaennip ( 깻잎).
O
O
OH
Bassoli, A., Borgonovo, G., Caimi, S., Scaglioni, L., Morini, G., Moriello, A.S., Di Marzo, V., De Petrocellis, L. (2009) Taste-guided
identification of high potency TRPA1 agonists from Perilla frutescens. Bioorganic Medicinal Chemistry, 17, 1636-1639.
Bassoli, A., Borgonovo, G., Morini, G., De Petrocellis, L., Schiano Moriello, A., Di Marzo, V. (2013) Analogues of perillaketone has
highly potent agonists of TRPA1 channel. Food Chemistry, 141, 2044-2051.
Extreme climate conditions
Poor economic developement and
hard physical work
Resistance to pain as a cultural
tradition
A “pain endurance” menu’?
Can a “TR(I)P enriched” diet help to raise
the threshold of pain endurance?
In regions with hard “environmental” conditions such as:
food is generally rich in TR(I)P active compounds
A.Bassoli-Cortona 13/06/2017
Alkaloids
TR(I)P active compounds
?
An extra mechanism to prevent/bear pain?
PAIN RESISTENCE THRESHOLD
PHYSICAL AND MENTAL FATIGUE
A.Bassoli-Cortona 13/06/2017
CONCLUSIONS:
Chemestetic properties in food and food plants can
indicate the presence of TRP active compounds;
TRP compounds are involved in pain perception and
their applications in this field have still to be fully
discovered;
TRP are involved in many sensing mechanisms and
have a lot of potential in medicine, pharmaceutical,
agriculture and food industry.
A.Bassoli-Cortona 13/06/2017 THANK YOU!