Small Molecule Agonists and Antagonists of TRPV4

Chapter 12

Small Molecule Agonists and Antagonists of TRPV4

Matthew A.J. Duncton*    Renovis, Inc. (a wholly owned subsidiary of Evotec AG), South San Francisco, California, USA
* Corresponding author:


Transient receptor potential vanilloid 4 (TRPV4), originally identified in 2000 as an osmosensor [13], is a member of the transient receptor potential (TRP) superfamily of ion channels. This superfamily, which consists of approximately 28 members, is divided into 7 subfamilies (TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, TRPV), based on sequence homology [4,5]. TRPV4 functions as a Ca2 +-permeable, nonselective cationic ion channel and is activated by numerous stimuli such as endogenous and exogenous small molecule ligands, temperature (> 27 °C), osmolarity, and phosphorylation by Src, protein kinase A (PKA), and protein kinase C (PKC) [13,611]. As documented by expression studies, TRPV4 seems to exhibit a broad tissue distribution [13,1219]. Since its discovery in 2000, TRPV4 has been implicated in numerous biological processes and medical conditions. Examples include osmolarity sensing and regulation [13], thermosensation and regulation [6,12,20], mechanosensation [20,21], bone formation and remodeling [2226], genetic disorders [24,25,2730], pain and inflammatory conditions [3138], bladder disorders [17,18], acute lung injury [3941], cardiovascular conditions [42], and metabolic disorders [43], among others. The purpose of this chapter is to highlight the most prominent small molecules that have been described as either agonists, or antagonists, of TRPV4. As such it should be regarded as an extension of two related articles published in 2010 and 2011 [44,45]. Because extensive coverage of the biology of TRPV4 is not included, readers are directed to some of the excellent reviews that already exist on this subject [4650].

TRPV4 Agonists

4α-PDD and Related Phorbol Esters

Phorbol esters, some of which are well-known tumor-promoters via activation of PKC, have also been described as agonists of TRP ion channels. For example, resiniferatoxin (RTX) 1 (Figure 12.1), a daphnane diterpene isolated from the latex of Euphorbia resinifera, is a potent activator of TRPV1, with a potency 103-105 times greater than pure capsaicin [51]. A search of related phorbol esters, by Nilius and coworkers, identified 4α-phorbol 12,13-didecanoate (4α-PDD) 2, as a potent activator of human and murine TRPV4 [52]. Significantly, 4α-PDD is not an activator of PKC (EC50 > 25 μM; [52]). A related phorbol ester, phorbol 12-myristate, 13-acetate (PMA) 3, was also found to be an activator of TRPV4, but was ca. 50 times less effective than 4α-PDD [52]. Unlike 4α-PDD, activation of PKC has been noted for PMA [52].


Figure 12.1 RTX, 4α-PDD, and PMA.

Structure-activity relationships (SARs) with 4α-PDD and relatives have also been investigated by the research groups of Nilius and Appendino [53]. This collaboration has revealed some fascinating SARs. For example, the 4-hydroxyl group was found to be essential for activity at TRPV4 because a deoxy congener of 4α-PDD, to give compound 4, was unable to activate TRPV4 (Figure 12.2). Studies with a series of 12,13-diesters, in which the carbon chain length was varied, resulted in the identification of a particularly potent agonist, 4α-phorbol 12,13-dihexanoate (4α-PDH) 5, which was approximately fivefold more active at TRPV4 than 4α-PDD. Interestingly, 4α-PDH and 4α-PDD represent the most potent 12,13-diesters found in this study because other variations in chain length resulted in a substantial reduction in TRPV4 activity. For example, phorbol dioctanoate 6 was essentially inactive at TRPV4, with an EC50 greater than 50 μM. It was also possible to remove one of the decanoate chains from 4α-PDD and still retain activity at TRPV4. For instance, mono-decanoate analog 7, in which the 12-ester group of 4α-PDD was removed, showed diminished relative efficacy to 4α-PDD, but with a comparable EC50 value. Finally, observations with 4α-lumiphorbol didecanoate (4α-LPDD) 8, a unique (2π-2π) photocyclized adduct of 4α-PDD, revealed differences between the interactions of their respective diterpene cores with the TRPV4 receptor.


Figure 12.2 Phorbol relatives of 4α-PDD.

Proposed Endogenous Agonists of TRPV4

A variety of endocannabinoids, such as anandamide 9 and 2-arachidonoylglycerol (2-AG) 10 (Figure 12.3), have been shown to activate TRPV4 via an indirect mechanism [7,8,52]. Hydrolysis of anandamide, or 2-AG, by their respective degrading enzymes, fatty-acid amide hydrolase, or mono-acyl glycerol lipase, yields arachidonic acid (AA) 11, which also serves as an indirect agonist of TRPV4 [8]. Through a thorough examination of the downstream metabolic fate of AA, Nilius and coworkers were able to establish that TRPV4 activation was occurring via the downstream metabolites, 5,6-eopxyeicosatrienoic acid (5,6-EET) 12, and 8,9-epoxyeicosatrienoic acid (8,9-EET) 13 [8]. These two epoxyeicosatrienoic acids are formed by the cellular oxidation of AA by a cytochrome P450 epoxygenase pathway, using the CYP 2C9 isoform. Thus, inhibitors of CYP 2C9 were able to abolish the activation of TRPV4 by AA [8]. The formation of 5,6-EET, or 8,9-EET, seems to be an important event for the activation of TRPV4 by osmotic cell swelling, as hypotonic cell swelling induces phospholipase-A2 activation and release of AA from the cell-membrane [7,8]. Inhibition of the P450 epoxygenase activity with miconazole (an inhibitor of CYP 2C9) strongly retarded the response of TRPV4 to hypotonic cell swelling. In contrast, the response of TRPV4 to other activating stimuli such as heat, or 4α-PDD, was unaffected by inhibition of P450 epoxygenase activity, indicating that these stimuli couple to TRPV4 by a pathway not involving AA.


Figure 12.3 Anandamide, 2-AG, AA, and endogenous agonists of TRPV4.

Dimethylallyl pyrophosphate (DMAPP) 14, an intermediate in the mevalonate metabolic pathway and found endogenously at nanomolar to micromolar concentrations, has also been identified as an agonist of TRPV4 (EC50 = 2.5 μM) [54]. At higher concentrations, DMAPP was also found to act as an antagonist at TRPV3 (IC50 = 10.4 μM). In vivo, DMAPP could promote inflammation in mice, the activity of which was blocked by codosing with the TRPV4 antagonists RN-1734 and HC-067047 [54]. Taken together with previous results on the action of mevalonate metabolites at TRP channels, a role for these metabolites as modulators of sensory TRP channels was proposed [54].

GSK1016790A and Relatives from GlaxoSmithKline

GlaxoSmithKline (GSK) has described some aspects of the medicinal chemistry and detailed in vivo pharmacology with a potent TRPV4 agonist, GSK1016790A 19 (Figure 12.4), and a series of structurally related relatives [5566]. The development of the medicinal chemistry was initiated with the identification of compound 15 as a submicromolar agonist of TRPV4 (EC50 = 0.7 μM). Previously, compound 15 was known to be a potent inhibitor of cathepsin K (IC50 = 2 nM) [65,66]. Through a series of detailed structure-activity studies, the chemistry group at GSK was able to abolish the activity at cathepsin K, while dramatically improving the functional activity at TRPV4. This resulted in the identification of a number of azepine (e.g., 16), diaminobutane (e.g., 17), or diaminopropane (e.g., 18) analogs as potent agonists of TRPV4, with favorable rat pharmacokinetic profiles [65,66]. Further refinement furnished GSK1016790A 19, which is based around a piperazine linker [6366]. Details of the in vitro and in vivo profile of GSK1016790A have been published [63,64]. For example, GSK1016790A is a potent agonist of TRPV4, with an EC50 of 3-5 nM at hTRPV4 and significant activity at bovine (EC50 = 1 nM), mouse (EC50 = 18.5 nM), rat (EC50 = 10 nM), and dog (EC50 = 1 nM) TRPV4 [63]. Significantly, the relative efficacy of GSK1016790A at TRPV4 is much greater than that of 4α-PDD. For example, the current density evoked by stimulation with GSK1016790A at 10 nM was over twice that recorded with 4α-PDD at a concentration of 10 μM [63]. In addition, GSK1016790A induces Ca2 + influx in HEK-293 cells, which do not express TRPV4 (EC50 = 50-100 nM). This calcium influx did not occur as a result of TRPV1 activation. In vivo, GSK1016790A induces bladder overactivity when infused directly into the bladders of mice. This effect was absent in TRPV4-knockout mice and led to the conclusion that TRPV4 plays a critical role in urinary bladder function [63]. Unfortunately, potent stimulation of the TRPV4 receptor has catastrophic side effects, as observed when GSK1016790A was administered intravenously to mouse, rat, or dog [64]. Such administration resulted in endothelial failure and circulatory collapse, resulting in a dose-dependent drop in blood pressure and death [64]. In TRPV4-knockout mice, the effect of GSK1016790A on blood pressure, or heart rate, was absent at doses greater than 10-fold the lethal dose in wild-type counterparts, indicating the effect was mediated by TRPV4 [64]. Further evaluation in dogs indicated that cardiac output was severely reduced on exposure to GSK1016790A, which was associated with decreased stroke volume and delayed bradycardia [64]. Overall, the catastrophic side effects associated with systemic exposure to a potent TRPV4 agonist, such as GSK1016790A, have resulted in a natural avoidance of such compounds as potential therapeutic agents.


Figure 12.4 GSK1016790A and related agonists from GlaxoSmithKline.

RN-1747 from Renovis

Vincent et al. at Renovis have identified an arylsulfonamide TRPV4 agonist, RN-1747 20 (Figure 12.5) [67]. RN-1747 (EC50 = 0.77 μM) activates human TRPV4 with a similar potency to 4α-PDD, but is somewhat less active than 4α-PDD against rat TRPV4 (EC50 = 4.1 μM), or mouse TRPV4 (EC50 = 4.1 μM). At higher concentrations, RN-1747 serves as an antagonist of TRPM8 (IC50 = 4 μM), but is selective against other TRP channels, such as TRPV1 and TRPV3 [67].


Figure 12.5 TRPV4 agonist, RN-1747.

Natural Products from Herbal Extracts

Bisandrographolide A (BAA) 21 (Figure 12.6), a natural product isolated from an extract of Andrographis paniculata, was shown to be an activator of mouse TRPV4 (EC50 = 0.79-0.95 μM) [68]. In addition, BAA was found to be relatively selective for TRPV4, with no activity noted at related TRP receptors, TRPV1, TRPV2, or TRPV3 [68].


Figure 12.6 Bisandrographolide A (BAA), a natural product active as a TRPV4 agonist.

TRPV4 Antagonists

Ruthenium red

Ruthenium red 22 (Figure 12.7) was one of the first small molecules to be described as an antagonist of TRPV4 [52]. The compound, a well-known cationic dye dating from the nineteenth century, functions as a pore blocker of the TRPV4 channel. Although ruthenium red has been utilized extensively as a probe of TRPV4 function, it suffers from poor selectivity because it also interacts with numerous ion channels and biological targets [44,45]. Such polypharmacology may be highly undesirable in a proof-of-concept probe compound, and ruthenium red has now been superseded by more potent and/or selective TRPV4 antagonists such as HC-067047, RN-1734, and GSK2193847, among others (see following sections).

Nov 18, 2017 | Posted by in PHARMACY | Comments Off on Small Molecule Agonists and Antagonists of TRPV4
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