L.R. Sadofsky et al. / Pharmacological Research 63 (2011) 30–36
35
of the substrate was mixed with the cysteine derivative and then
the reaction progress monitored by proton NMR over a period of
pared to acrylic acid NHS ester and acrolein. This confirmed the
data seen with the TRPA1–HEK cells. Hydrocinnamic acid NHS, the
only compound tested in this model which reacts solely by direct
addition, failed to produce significant nerve depolarization at the
highest concentration tested (1 mM). Poor aqueous solubility of this
compound prevented the use of higher concentrations, which are
needed for this method, which may have evoked responses. This
problem was also observed with cinnamic acid NHS ester.
The activity of formalin, an agent known to induce hyperalge-
sia, which can only react via direct addition at the carbonyl carbon,
confirms the importance of this mechanism of TRPA1 activation
and has consequences for our understanding of this process. If ago-
nists work by this mechanism then the activity of acrolein and
cinnamaldehyde may be triggered by fast and readily reversible
addition of cysteine residues to their carbonyl carbon, and not nec-
essarily by the conjugate addition process that has been inferred
by others [5,6]. Addition of a thiol to the carbonyl carbon of a reac-
tive aldehyde is fast, but forms an unstable hemithioacetal and so
the reverse process can also occur readily. Conjugate addition how-
ever is a relatively slower process, but produces a much more stable
-thioaldehyde. Thus activation of TRPA1 by this newly proposed
mechanism is compatible with our initial observation of brief trig-
gering of the cough reflex by acrolein and cinnamaldehyde [2], and
may be the principle mechanism of the sensing of these environ-
mental irritants in vivo.
1
20 min (Fig. 2B). The alkenyl signal at ı 7.91, arising from the
hydrogen atoms labelled Ha (Fig. 2A), was free from overlap with
other signals in the spectrum. If the conjugate addition process
was occurring then this signal would have disappeared but been
replaced by new alkyl signal at a much lower chemical shift (<ı 6.0).
The disappearance of the signal for Ha (starting material, Fig. 2A)
was duly observed, and was replaced by that at ı 7.54 (product).
This confirmed the direct addition process was occurring, not con-
jugate addition and was therefore in agreement with the literature
precedence [10]. The final ratio of starting material to product did
not reach the theoretical 3:1, undoubtedly due to slow formation
of the cysteine disulfide.
We have shown here that these TRPA1 agonists do indeed react
with cysteine residues via direct addition and taken together with
the results of Hinman et al. [5] and Macpherson et al. [6] which
demonstrated that removal of specific cysteine residues ablated
agonist responses, the results suggest that these compounds are
activating TRPA1 via covalent cysteine modification.
Compounds such as acrylamide, acrylic acid, and cinnamic acid,
are effectively only able to react with cysteine residues via con-
jugate addition. These however failed to activate TRPA1 up to
concentrations of 300 M. Our NMR experiment, backed up by rele-
vant literature [9], showed however that cysteine derivatives react
relatively readily with acrylamide in a conjugate fashion when in
a protic solvent, and yet the pharmacology does not reveal a cor-
respondingly robust physiological response. In contrast acrolein,
methyl vinyl ketone, mesityl oxide, and cinnamaldehyde, which
can react via either conjugate or reversible direct addition, caused
TRPA1 activation in a concentration dependent manner. These
results then may indicate that with reactive aldehydes such as
acrolein, it is the reversible direct addition which is responsible
for the channel activation in a similar fashion to formaldehyde.
This would then explain our observation that TRPA1 agonists such
as cinnamaldehyde acted in a manner compatible with reversible,
competitive agonism. Acrylic acid and cinnamic acid NHS esters
also caused TRPA1 activation in a concentration dependent man-
ner, in which the reaction with the cysteine residues occur via an
essentially irreversible direct addition. Pre-incubation of cells with
increasing acrylamide concentrations failed to inhibit acrylic acid
NHS ester responses confirming that acrylamide does not interact
with the TRPA1 cysteines involved in the acrylic acid NHS ester
evoked responses (data not shown).
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