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. 2014 May 12;9(5):e97005.
doi: 10.1371/journal.pone.0097005. eCollection 2014.

Transient receptor potential ankyrin 1 receptor activation in vitro and in vivo by pro-tussive agents: GRC 17536 as a promising anti-tussive therapeutic

Indranil Mukhopadhyay  1 , Abhay Kulkarni  1 , Sarika Aranake  1 , Pallavi Karnik  1 , Mahesh Shetty  1 , Sandeep Thorat  1 , Indraneel Ghosh  1 , Dinesh Wale  1 , Vikram Bhosale  1 , Neelima Khairatkar-Joshi  1
Affiliations

Transient receptor potential ankyrin 1 receptor activation in vitro and in vivo by pro-tussive agents: GRC 17536 as a promising anti-tussive therapeutic

Indranil Mukhopadhyay et al. PLoS One. .

Abstract

Cough is a protective reflex action that helps clear the respiratory tract which is continuously exposed to airborne environmental irritants. However, chronic cough presents itself as a disease in its own right and despite its global occurrence; the molecular mechanisms responsible for cough are not completely understood. Transient receptor potential ankyrin1 (TRPA1) is robustly expressed in the neuronal as well as non-neuronal cells of the respiratory tract and is a sensor of a wide range of environmental irritants. It is fast getting acceptance as a key biological sensor of a variety of pro-tussive agents often implicated in miscellaneous chronic cough conditions. In the present study, we demonstrate in vitro direct functional activation of TRPA1 receptor by citric acid which is routinely used to evoke cough in preclinical and clinical studies. We also show for the first time that a potent and selective TRPA1 antagonist GRC 17536 inhibits citric acid induced cellular Ca(+2) influx in TRPA1 expressing cells and the citric acid induced cough response in guinea pigs. Hence our data provides a mechanistic link between TRPA1 receptor activation in vitro and cough response induced in vivo by citric acid. Furthermore, we also show evidence for TRPA1 activation in vitro by the TLR4, TLR7 and TLR8 ligands which are implicated in bacterial/respiratory virus pathogenesis often resulting in chronic cough. In conclusion, this study highlights the potential utility of TRPA1 antagonist such as GRC 17536 in the treatment of miscellaneous chronic cough conditions arising due to diverse causes but commonly driven via TRPA1.

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Conflict of interest statement

Competing Interests: All the authors are employees of Glenmark Pharmaceuticals Ltd. IM has one patent issued (Thienopyrimidinedione derivatives as trpa1 modulators; Patent No. US8507503), AK has one patent filed (Application No. 3067/MUM/2013) and NKJ has one patent issued (Thienopyrimidinedione derivatives as trpa1 modulators; Patent No. US8507503) and one filed (Application No. 3067/MUM/2013), which is broadly relevant to the work. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. However, the authors will not be able to disclose the structure and/or share compound GRC 17536, which is currently under clinical development but will be happy to spare other compounds as mentioned above.

Figures

Figure 1
Figure 1. Citric acid-induced Ca+2 influx in (A) CCD19-Lu cells, (B) A549 cells and (C) hTRPA1/CHO cell line.
Each point in the graph is an average ± SEM of at least two independent experiments.
Figure 2
Figure 2. Blockade of citric acid-induced Ca+2 influx by TRPA1 and TRPV1 antagonists.
(A–B) Inhibition of citric acid-induced Ca+2 influx by GRC 17536, compound 7 and GRC 6211 in CCD19-Lu (A) and A549 (B) cells. (C) Inhibition of citric -induced Ca+2 influx by GRC 17536, compound 7 and GRC 17770 in hTRPA1/CHO cells. Each point in the graph is an average ± SEM of at least two independent experiments.
Figure 3
Figure 3. Effect of TLR4, TLR7 and TLR8 ligand on TRPA1 activation.
(A) Effect of LPS on TRPA1 agonist (crotonaldehyde, H2O2 and 15d-PGJ2)-mediated 45Ca+2 uptake in hTRPA1/CHO cells. (B) Effect of loxoribine and ssRNA40 on AITC mediated 45Ca+2 uptake in hTRPA1/CHO cells. (C) Effect of LPS, loxoribine and ssRNA40 on AITC mediated 45Ca+2 uptake in A549 cells. (D) LPS, loxoribine and ssRNA40 enhance TRPA1 expression. RT-PCR was performed with mRNA isolated from hTRPA1/CHO cells. Each histogram is the mean ± SE of at least two independent experiments.
Figure 4
Figure 4. Blockade of TLR agonist-mediated enhanced 45Ca+2 uptake by TRPA1 antagonists in (A) hTRPA1/CHO cells and (B) A549 cells.
Each histogram is the mean ± SE of at least two independent experiments.
Figure 5
Figure 5. Effect of GRC 17536 on citric acid (0.4 M) induced cough response in male guinea pigs.
See this image and copyright information in PMC

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Grants and funding

The work was funded by Glenmark Pharmaceuticals Ltd. and its subsidiary. The funder is a legal entity and not a natural person, and hence has no role in the design, data collection, analysis or preparation of the manuscript.

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