Species-Specific Pharmacology of Trichloro(Sulfanyl)ethyl Benzamides as Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists

Abstract

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.

Mouse Monoclonal Antibodies to Transient Receptor Potential Ankyrin 1 Act as Antagonists of Multiple Modes of Channel Activation

“…In addition to reactive chemical activators, cold temperature has been shown to activate TRPA1 in recombinant expression systems (Story et al, 2003;Corey et al, 2004;Story and Gereau, 2006;Klionsky et al, 2007). Furthermore, recent studies unequivocally showed that cold indeed activates TRPA1 in calcium imaging experiments as well as in single channel recordings (Sawada et al, 2007).…”

“…Generation of Chinese hamster ovary (CHO) cell lines stably expressing human transient receptor potential ankyrin 1 (hTRPA1), rat transient receptor potential ankyrin 1 (rTRPA1), or human transient receptor potential melastatin 8 (hTRPM8) and their functional characterizations were previously described (Klionsky et al, 2007;Gavva et al, 2012). To construct the hTRPA1-FLAG sequence, a DNA fragment encoding the FLAG tag (DYKDDDDK) was inserted into the predicted extracellular loop 1 of hTRPA1 at amino acid 756.…”

“…The EC 90 concentration (80 mM) of AITC (based on previous concentration response studies ;Klionsky et al, 2007) was used for activation of TRPA1 in these assays. The addition of the TRPA1 antagonist mAbs 2B10 and 2D1 to the cells inhibited 45 Ca 21 uptake in a concentration-dependent manner ( Fig.…”

“…CHO cells expressing human TRPA1 or TRPM8 were used in the agonist-induced 45 Ca 21 uptake assay (Klionsky et al, 2007) to identify TRPA1 antagonist mAbs. Twenty-four hours before the assay, cells were seeded in Cytostar 96-well plates (GE Healthcare Life Sciences) at a density of 3.0 Â 10 4 per well and TRPA1 channel expression was then induced with 0.5 mg/ml tetracycline.…”

TRP Channels: Their Function and Potentiality as Drug Targets

“…AMG5445 inhibits human TRPA1, but activates rat TRPA1 [ 70 ]. The pharmacological profi le of the human and rhesus monkey TRPA1 is relatively distinct from mouse and rat TRPA1 [ 94 ].…”