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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 :


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


Ca 2+ release

Activation

(conformational change?)

Stimulus

(physical or

chemical)

How does a TRP receptor work?

signal


How many TRP receptors do we have?

The phylogenetic tree of TRP receptors

D.E. Clapham, Nature, 426, 517-524, 2003


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


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

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


http://ki.se/?l=en

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 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


1957: Nicholas Jancsò

hypothesised the existence of a

specific receptor

TRPV1


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 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.


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.


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


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)


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)


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


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


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.


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

http://www.stockfood.it/results.asp?inline=true&image=308135&wwwflag=3&imagepos=24

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.


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


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


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.


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


Alkaloids

TR(I)P active compounds

?

An extra mechanism to prevent/bear pain?

PAIN RESISTENCE THRESHOLD

PHYSICAL AND MENTAL FATIGUE


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!

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