Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists

Agonists of TRPA1 such as mustard oil and its key component AITC cause pain and neurogenic inflammation in humans and pain behavior in rodents. TRPA1 is activated by numerous reactive compounds making it a sensor for reactive compounds in the body. Failure of AITC, formalin and other reactive compounds to trigger pain behavior in TRPA1 knockout mice, as well as the ability of TRPA1 antisense to alleviate cold hyperalgesia after spinal nerve ligation, suggest that TRPA1 is a potential target for novel analgesic agents. Here, we have characterized CHO cells expressing human and rat TRPA1 driven by an inducible promoter. As reported previously, both human and rat TRPA1 are activated by AITC and inhibited by ruthenium red. We have also characterized noxious cold response of these cell lines and show that noxious cold activates both human and rat TRPA1. Further, we have used CHO cells expressing human TRPA1 to screen a small molecule compound library and discovered that 'trichloro(sulfanyl)ethyl benzamides' (AMG2504, AMG5445, AMG7160 and AMG9090) act as potent antagonists of human TRPA1 activated by AITC and noxious cold. However, trichloro(sulfanyl)ethyl benzamides' (TCEB compounds) displayed differential pharmacology at rat TRPA1. AMG2504 and AMG7160 marginally inhibited rat TRPA1 activation by AITC, whereas AMG5445 and AMG9090 acted as partial agonists. In summary, we conclude that both human and rat TRPA1 channels show similar AITC and noxious cold activation profiles, but TCEB compounds display species-specific differential pharmacology at TRPA1

Effect of human TRPA1 antagonists on CHO cells stably expressing rat TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Concentration-response curves generated in aequorin-readout assay Note, AMG9090 and AMG5445 acted as partial agonists. Each point in the graph is presented as percent of AITC response and is an average ± SEM of an experiment conducted in triplicate. Representative traces of inward currents induced by AITC, AMG9090 and AMG5445 are shown in , , and respectively. AITC-induced increase in intracellular calcium in CHO cells expressing rat TRPA1 in the absence (100%) or presence of different concentrations of AMG2504 and AMG 7160. Representative current traces induced by AITC in the presence of AMG2504 and AMG 7160 are shown in and , respectively

Trichloro(sulfanyl)ethyl benzamides inhibit cold temperature (4°C) induced Cauptake in CHO cells stably expressing human TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Concentration-response curves were generated utilizing Cauptake assays as described under . Each point in the graph is an average ± SEM of an experiment conducted in duplicate

Characterization of CHO cell lines stably expressing human and rat TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> FLASH luminometer was used to measure concentration-dependent AITC-induced increase in intracellular calcium, and concentration-dependent inhibition of AITC (80 μM) activation by ruthenium red in CHO cells expressing human and rat TRPA1. Each point in the graph is an average ± SEM of an experiment conducted in triplicate. Maximum response of AITC (80 μM) was normalized to 100%. Cold activation profiles of CHO cells expressing human and rat TRPA1 were characterized utilizing cold-induced Cauptake assay. Cauptake by CHO cells and CHO cells transfected with human TRPA1 in response to stimulation with temperatures between 3.5 and 25°C. Cold temperature (4°C) activation of un-induced and tetracycline-induced CHO cells transfected with either human or rat TRPA1. Concentration-dependent inhibition of cold (4°C) activation by ruthenium red in CHO cells expressing human and rat TRPA1. Each point in the graph is an average ± SEM of an experiment conducted in duplicate

Trichloro(sulfanyl)ethyl benzamides inhibit AITC (80 μM) induced inward currents in CHO cells stably expressing human TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Representative traces of inward currents evoked by AITC in the absence or presence of AMG9090 , AMG5445 , AMG2504 , and AMG7160 are shown. ICvalue for each compound was determined from their concentration-dependent inhibition of AITC-induced currents using a PatchXpress 7000A workstation

Trichloro(sulfanyl)ethyl benzamides inhibit AITC (80 μM) induced increase in intracellular calcium and inward currents in CHO cells stably expressing human TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Effect of AMG9090 , AMG5445 , AMG2504 , and AMG7160 on AITC-induced increase in intracellular calcium in CHO cells expressing human TRPA1 measured in an aequorin-readout assay. Open circles represent the response of cells to the compound itself in the absence of agonist. Each point in the graph is an average ± SEM of an experiment conducted in triplicate