TRPA1, what a therapeutic target for pain!

At the end of the first quarter of 2023, many pharmaceutical companies disclosed major adjustments to their R&D pipelines in their quarterly reports. Eli Lilly also announced they will give up on three clinical trials which include TRPA1 antagonist. Eli Lilly’s TRPA1 antagonist was acquired from Hydra Biosciences in 2018 and is mainly used for the treatment of chronic pain syndromes. What is TRPA1? Apart from Eli Lilly, which other pharmaceutical companies work in this pipeline, and what is the current progress?

1. What is TRPA1?

The transient receptor potential (TRP) protein family is a class of non-selective cation channels located on the cell membrane. According to their amino acid sequence homology, mammalian TRP channels can be divided into seven subfamilies, including TRPC, TRPV, TRPM, TRPP, TRPML, TRPA, and TRPN [1]. Transient receptor potential ankyrin 1 (TRPA1, also known as ANKTM1), the only member of the TRPA subfamily, is a Ca2+-permeable non-selective cation channel that plays an important role in different types of pain.

TRPA1 is abundantly expressed in primary sensory neuron subpopulations of the trigeminal ganglion, vagal ganglion, and dorsal root ganglion, as well as in non-neuronal cells such as macrophages, dendritic cells, T lymphocytes, neutrophils, and mast cells [2]. In addition, emerging evidence has shown that TRPA1 is also expressed in peptidergic and non-peptidergic neurons. Peptidergic neurons are rich in neuropeptides CGRP and SP and neurotrophic factor receptor TrkA; non-peptidergic neurons are co-expressing purinergic receptors P2X3, Neurturin, Artemin, Mrg GPCR family, as well as GFRα1 and GFRα2 in the GDNF receptor family [3]. In recent years, the expression of TRPA1 has also been found on some non-neural cells, such as Nell hair cells, vascular endothelial cells, dental pulp fibroblast keratinocytes, pancreatic islet cells, etc. [4]

2. What is the structure of TRPA1?

The TRPA1 gene was first cloned in 1999 from lung fibroblasts. TRPA1 in humans consists of 1119 amino acids with a relative molecular mass of about 127kDa, located on human chromosome 8 q13. As shown in Figure 1, the TRPA1 transmembrane protein consists of 6 transmembrane domains (S1-S6) and 1 pore canal, and its N-terminal and C-terminal parts are both inside the cell. S1 and S2 domains form the extracellular ring structure, and the hydrophilic regions of the transmembrane domains of S5 and S6 form the pore canal. The long NH2 terminus of the TRPA1 protein contains the most prevalent ankyrin repeat domain (ARD) in the TRP superfamily, including 14-16 ankyrin repeats. Each ARD is composed of an α-helix- β-turn-α-helix motif composition [5] and may be involved in protein-protein interactions, such as binding with phospholipase C (PLC) and calcium (Ca2+). ARD is linked to TM1 through the pre-S1 region, which contains some cysteine residues (such as Cys621, Cys641, and Cys665) that are critical for the activation of TRPA1 by electrophilic agonists. There is an EF-hand binding domain at the N-terminus, which can increase intracellular calcium ions. TRPA1 can be activated by many exogenous small organic molecules such as horseradish, cinnamaldehyde, cannabis, allicin, mustard, and proton, but has no response to menthol.

Figure1. the structure of TRPA1[6]

*The picture is derived from Moccia F et al.

3. What is the mechanism of TRPA1 involved in pain?

The TRPA1 channel protein was discovered by Professor Ardem Patapoutian, winner of the 2021 Nobel Prize in Physiology or Medicine. It is one of the non-selective ligand-gated cation channels involved in sensing noxious stimuli and conducting noxious signals. TRPA1 can be activated by exogenous stimuli in three different ways:

  • Exogenous stimuli activate protein kinase C (PKC) through G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), which modulates the activity of the TRPA1 channel;
  • Low-molecular-weight organic compounds and endogenous esters act as ligands for TRPA1 to activate the channel;
  • Temperature changes and mechanical stimuli act directly on TRPA1 channels to promote their opening and activation.

Activation or sensitization of TRPA1 channels increases Ca2+ influx, promotes the neurogenic inflammatory response, and releases neuropeptides such as substance P (SP), neurokinin A (NKA), and calcitonin-related peptide (CGRP).

Multiple studies have demonstrated that TRPA1 plays an important role in mediating long-term hypersensitivity to thermal, chemical, and mechanical stimuli in models of nociceptive and neuropathic pain. The role of TRPA1 in inflammatory nociception was first demonstrated by McNamara CR et al. This study indicated that formalin-induced nociceptive responses were reduced in rat and mouse paws following pharmacological antagonism or gene deletion of the TRPA1 channel [8]. Moreover, acute inhibition of TRPA1 reduced cold and mechanical hypersensitivity associated with persistent inflammation in carrageenan- and complete Freund’s adjuvant-induced animal models [9]. In addition, TRPA1 has also been shown to contribute to peripheral and central neuropathic pain. TRPA1 antagonists reduced mechanical allodynia and hypersensitivity in a rodent model of streptozotocin-induced diabetic neuropathy [10] [11].

4. What is the current progress of drugs targeted TRPA1?

Ruthenium red and Gentamicin were the first TRPA1 channel antagonists reported in the literature, and these nonspecific channel blockers have been replaced by more selective TRPA1 antagonists. TRPA1, as an analgesic target of ion channels, does not have the risk of addiction and abuse of opioids. Because it is upstream of the pain signaling pathway, drugs targeted TRPA1 will not affect the body’s sensory patterns while blocking pain signals. TRPA1 is superior to other analgesic targets with the feature of low-expression levels in the central nervous system and heart, which further reduces the risk of the central nervous system and cardiac side effects.

4.1 CB 189625

CB 189625, also known as CB-625, is a novel small molecule antagonist of the human TRPA1 channel. Different from the mechanism of other drugs, CB 189625 acts on peripheral pain fibers and blocks peripheral TRPA1 channels to relieve surgery-induced and inflammation-mediated pain in animal models. In 2012, Cubist Pharmaceuticals and Hydra Biosciences announced a joint phase I clinical trial of the TRPA1 antagonist CB 189625. Since then, the CB 189625 trial was terminated due to poor pharmacokinetic properties. In 2015, Cubist Pharmaceuticals was acquired by Merck.

4.2 GRC 17536

GRC 17536, also known as ISC 17536, is also an orally small molecule antagonist of the TRPA1 channel, which is currently in clinical development by Glenmark Pharmaceuticals. Preclinical studies have certified the effectiveness of GRC 17536 in animal models of neuropathic and inflammatory pain, including diabetic peripheral neuropathic, osteoarthritis, postoperative, and chemotherapy-induced pain. And these results also validated the TRPA1 blockade potential utility of medications in therapeutic pain management.

In September 2014, Glenmark announced that it had achieved positive results from a Phase IIa proof-of-concept study in painful diabetic peripheral neuropathy, with statistically significant and clinically relevant responses in a subgroup of patients with moderate to severe diabetic neuropathic pain. GCR 17536 is currently suspended for clinical use by FDA, and the official materials are expected to resume clinical trials in the second half of 2019.

4.3 ODM108

In April 2015, Orion announced that TRPA1 antagonist ODM108 had entered phase I clinical trials for neuropathic pain in the Netherlands. However, in July 2016, the trial was discontinued after completion of the second part of the phase I clinical trial in 2016 due to unfavorable pharmacokinetic properties.

4.4 LY-3526318

As mentioned before, LY-3526318 was originally developed by Hydra Biosciences, and in 2018, Lilly started to promote clinical trials. LY-3526318, also known as LY356318, is a small molecule antagonist of TRPA1. On October 13, 2022, Lilly completed Phase II clinical trials for osteoarthritis, chronic low back pain, and neuropathic pain in the United States and Puerto Rico. On October 14, 2022, Lilly plans to conduct a Phase I safety and pharmacokinetic trial in Japanese healthy volunteers. But on January 9, 2023, Eli Lilly withdrew a phase I trial before recruiting healthy volunteers (PO) in Japan because of a commercial decision.

Despite numerous studies of TRPA1 across the pharmaceutical industry over the years, only GRC 17536 has reached the endpoint of phase II clinical trial. Both CB 189625 and ODM108 were terminated due to poor pharmacokinetics, mainly due to poor solubility and low bio-availability. In China, currently, only Leado Pharma’s TRPA1 antagonist LDS has the fastest progress, and the IND declaration has been completed. LDS has shown potent analgesic activity in a variety of classic pain pharmacodynamic models, and it is significantly better than the first-line treatment in clinical use.

5. The full-length TRPA1 nanodisc protein supplied by DIMA Biotechnology

As you know, TRPA1 is a six-pass transmembrane channel protein. In order to develop good antibody-based or small molecule drugs for multipass transmembrane proteins, it is crucial and difficult to obtain an active full-length transmembrane protein. DIMA Biotechnology has developed the full-length human TRPA1 protein using its proprietary, Synthetic Nanodisc membrane protein expression technology platform.

Unlike most MSP (Membrane Supporting Protein) Nanodiscs, DIMA’s Synthetic Nanodisc is based on a mammalian cell expression system, and the final protein is produced directly from the original cell membranes. In this process, the synthetic polymers used have a dual function. First, it dissolves cell membranes, similar to detergents, and then uses natural cellular phospholipids to form nanodisc structures around membrane proteins. The final nanodisc proteins are soluble in buffer which can be purified by affinity purification.

At present, DIMA Biotechnology has developed hundreds of membrane proteins using the Nanodiscs full-length membrane protein platform. Click to view all of the nanodisc proteins supplied by DIMABIO.

Human TRPA1 full length protein-synthetic nanodisc (FLP100033)

[Left] TRPA1 nanodisc protein can react with anti-Flag monoclonal antibody. the EC50 for anti-Flag monoclonal antibody binding with TRPA1-Nanodisc is 7.433ng/ml.
[Right] Human TRPA1-Nanodisc, Flag Tag on SDS-PAGE


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