TRPV4 and Drug Discovery

Fabien Vincent* and Matthew A. J. Duncton*

* Corresponding authors:

Fabien VincentPfizer, Inc. 558 Eastern Point Rd, Groton, 06340, CT.Tel: (650)-619-0722fabien_vincent_us@yahoo.com

Matthew A. J. DunctonFormerly with Renovis, Inc. (a wholly owned subsidiary of Evotec AG), Two Corporate Drive, South San Francisco, 94080, CA.Tel: (917) 345-3183mattduncton@yahoo.com

1

Abstract

Transient Receptor Potential Vanilloid 4 (TRPV4) was first identified in 2000 as

an osmolarity sensor. Further investigations rapidly revealed this ion channel to be a

polymodal receptor with additional activating or modulating stimuli including warm

temperatures, endogenous lipids and phosphorylation. The broad tissue and cell type

distribution of TRPV4, coupled with its varied activation profile, lead to a wide variety of

physiological roles. These include sheer stress detection in blood vessels, osteoclast

differentiation control in bone along with temperature monitoring in skin keratinocytes

and osmolarity sensing in kidneys. Recent work also implicated TRPV4 mutations in

multiple genetic disorders such as brachyolmia and Charcot-Marie Tooth disease 2C.

Characterization of its roles in disease states naturally led to a rising interest in the

modulation of TRPV4 for therapeutic purposes. Therapeutic areas of interest are diverse

and include several with significant unmet medical needs such as inflammatory and

neuropathic pain, bladder disfunctions as well as mechanical lung injury. Herein we

review the roles of TRPV4 in pathologies and summarize the progress made in

identifying small molecule modulators of its activity for target validation and therapeutic

purposes.

2

Introduction

The TRP ion channel superfamily is composed of the TRPA, TRPC, TRPM,

TRPN, TRPP and TRPV families, with a total of 28 members in mammals [1, 2]. TRPV4

belongs to the TRPV (vanilloid) channel family, itself subdivided into two subfamilies

with non selective cation permeable channels (PCa/PNa≤10) highly sensitive to temperature

changes (TRPV1-4) and Ca2+ selective channels (PCa/PNa>100) insensitive to temperature

variations (TRPV5-6) [3-5]. TRPV1, the canonical member of this family, was first

identified as the receptor of the active ingredient of chili peppers, capsaicin [6].

Additional studies uncovered other activating modalities such as noxiously high

temperatures (> 42ºC), acidity (< pH5), and lipids (e.g. anandamide) [7, 8]. As activation

by a given stimulus will modulate the sensitivity of the ion channel to other inputs,

TRPV1 was shown to function as a polymodal sensor, integrating a variety of activating

signals to produce a resulting cellular output [8].

TRPV4 is closely related to the TRPV1 receptor, sharing with it 40.9% of its

sequence. The human gene is located on chromosome 12q23-q24.1, with five proposed

splice variants produced from 12 exons [9, 10]. The 871 amino acids of full length

hTRPV4 define six transmembrane domains, with the pore region being located between

TM5 and TM6, as well as six ankyrin repeats near the N terminus, a PRD domain close

to the first ankyrin domain and putative calmodulin domains near the C terminus [4, 5, 9].

As with TRPV1, TRPV4 is believed to form homo-tetramers based on cryo-electron

microscopy studies using detergent-solubilized rat TRPV4 [4, 11]. Helpfully for drug

discovery work, rat and mouse sequences exhibit 95% identity with their human

counterpart [4, 5, 9]. The seminal studies which led to the discovery of TRPV4 revealed

3

its modulation by osmolarity [12-14]. Additional stimuli were soon identified, including

warm temperatures (>27°C) [15], phosphorylation by Src, PKC and PKA [16-18], and

endogenous lipids [4, 19, 20].

TRPV4 displays a wide expression pattern with its presence being documented in

kidney, lung, brain, dorsal root ganglia (DRG), bladder, skin, vascular endothelium, liver,

testis, fat, inner ear, pancreas, cornea and heart [4, 13, 14, 21-29]. Correspondingly,

TRPV4 is found in a variety of cell types, both excitable and non-excitable, including

peripheral, hippocampal and subfornical organ neurons, renal epithelial and urothelial

cells, airway smooth muscle cells, chondrocytes, osteoblasts and osteoclasts, smooth

muscle cells of the aorta, insulin-producing cells, astrocytes and others [3, 4, 12, 23,

30-40].

This widespread expression pattern, coupled with multiple activating modalities,

leads to a diversity of biological roles for TRPV4. Detailing these is beyond the scope of

this article - the reader is referred to several recent reviews [4, 9, 41] - while some areas

of interest for drug discovery will be expanded upon. Briefly, functional studies

investigating TRPV4 functions were conducted using a range of tools including small

molecule agonists and antagonists, antisense oligodeoxynucleotides, KO mice, and

specific genetic mutations linking TRPV4 to rare genetic disorders [12, 15, 26, 30, 33,

42-47]. Using these, TRPV4 was demonstrated to play a role in osmolarity sensing and

regulation in the CNS [12-14, 30, 48], thermosensation and possibly thermoregulation

[15, 21, 49], bladder function [43, 47], bone formation and remodeling [38, 40, 45, 46,

50], and mechanosensation in the vascular endothelium [37, 51].

4

A number of small molecules have been described as being either agonists or

antagonists of TRPV4. As a detailed discussion of every such compound is not possible

here, the reader is directed towards an earlier review providing a more detailed focus on

this topic [52]. This mini review will nonetheless contain a succinct account of the major

TRPV4 modulators, together with some applications of these compounds in vivo.

Areas of interest for drug discovery

Inflammatory and neuropathic pain

TRPV4 is expressed in peripheral nociceptive neurons (DRG and trigeminal (TG)

neurons), with data suggesting its transport to peripheral nerve endings [32, 33]. As

TRPV4 was originally identified as an osmosensor, early work using TRPV4 KO

investigated whether this channel was also involved in mechanosensation. These studies

provided the first evidence of TPRV4’s involvement in nociception with KO mice

displaying an increase in mechanical nociceptive threshold while baseline mechanical

sensitivity remained unaffected [12, 44].

Using complementary approaches to lower TRPV4 activity, TRPV4 KO mice and

TRPV4 knock down in rats using antisense oligodeoxynucleotides (AS ODN),

Alessandri-Haber, Levine and coworkers have produced a convincing body of work

documenting the involvement of TRPV4 in pain under pathological conditions [33, 34,

53-58]. Prostaglandin E2, a known inflammatory mediator, was originally observed to

sensitize this ion channel to both hypotonic and hypertonic media, conditions relevant to

diabetes and asthma for example [33, 56, 59]. This finding was later extended through the

use of several additional inflammatory mediators, leading to the discovery that increased

5

cAMP levels as well as PKA and PKC activation were necessary for TRPV4-mediated

mechanical hyperalgesia [54]. Further studies investigated its connection with protease-

activated receptor 2 (PAR2), a GPCR co-expressed with TRPV4 on a subset of primary

afferent neurons. The activation of PAR2 by proteases during inflammation sensitizes

TRPV4 through the stimulation of PLC, PKA, PKC and PKD [60]. Several groups have

now confirmed the link between PAR2 activation and TRPV4-mediated mechanical

hyperalgesia and release of nociceptive peptides substance P and CGRP [60-62]. Besides

its modulation by PAR2, TRPV4 was documented to be highly present on colonic

afferent neurons in the same studies and its role in visceral pain was demonstrated using

both TRPV4 KO and AS ODN approaches [62, 63]. Interestingly, PAR4, a relative of

PAR2, was recently documented to antagonize PAR2 and TRPV4-induced hyperalgesia

in response to colorectal distension [64].

Peripheral nerve damage from diverse etiologies can lead to allodynia or

hyperalgesia in patients. Alessandri-Haber, Levine and coworkers demonstrated that

TRPV4 mediates mechanical hyperalgesia in multiple rodent models of painful peripheral

neuropathy [34, 55]. The wide range of reagents used for nerve injury in these models

(paclitaxel, vincristine, streptozocin, ddC and alcohol) suggests a potentially broad role

for TRPV4 in neuropathic pain [55]. Mechanistic investigations revealed a requirement

for both Src tyrosine kinase and 21 integrin in TRPV4-mediated mechanical

hyperalgesia [55]. TRPV4 expression levels were observed to increase in a rat model of

chronic constriction of the DRG (CCD). TRPV4 AS ODN reversed this increase and

partially blocked the observed mechanical allodynia while leaving baseline nociception

unaffected [32]. Additionally, the PAR2-TRPV4 relationship was found recently to

6

extend beyond inflammatory conditions and to include Paclitaxel-induced mechanical

and thermal hyperalgesia [65].

Overall, data appear to suggest that TRPV4 may selectively mediate (mechanical)

nociception under pathological conditions, without altering baseline mechanosensation

[54]. TRPV4 antagonists may therefore prove therapeutically useful in the treatment of

inflammatory and neuropathic pain, potentially including visceral pain related to Crohn’s

Disease and inflammatory bowel syndrome.

Bladder dysfunction

TRPV4 is expressed in the bladder urothelium (both basal and intermediate cells),

in bladder smooth muscle cells, as well as in the urothelium cells lining the renal pelvis,

ureters and urethra [26, 27, 43, 66, 67]. Importantly, both mRNA and protein were

documented to be present in the urothelium, with increases in intracellular calcium being

observed in presence of well known TRPV4 activating modalities such as 4PDD,

GSK1016790A and hypotonicity [26, 27, 43]. This pharmacological response was

however reduced (hypotonicity) or absent (4PDD and GSK1016790A) in TRPV4 KO

cells stimulated in the same fashion [27, 43].

Mice lacking TRPV4 displayed a perturbed urine-voiding pattern, with longer

intervoiding intervals, larger voided volumes, fewer productive voiding contractions and

a disturbed spatial distribution of urine [27]. This data is consistent with TRPV4 ablation

leading to higher bladder filling and delayed micturition [68]. Conversely, use of TRPV4

agonists 4PDD in rats (but not mice) and GSK1016790A led to increased intravesical

pressure and bladder overactivity, respectively [26, 43]. Notably, this effect of

GSK1016790A was not observed in KO mice. More recently, the decrease in bladder

7

capacity and voided volume prompted by GSK1016790A infusion were negated by the

application of selective TRPV4 antagonist RN-1734 [69].

Other TRP channels are expressed in the bladder and are known to contribute to

the regulation of its function [68, 70-72]. TRPV1 expression levels in TRPV4 WT and

KO mice were found to be similar [27]. Conversely, no significant change in TRPV4

expression was observed in TRPV1 KO animals suggesting these ion channels do not

compensate for each other [27, 68].

Following the activation of the urothelium mechanosensory system, ATP is

released to activate P2X3 and P2X2/3 purinergic channels on sensory afferents. This

pressure-initiated ATP release is decreased in TRPV4 null mice, a result consistent with

the postulated mechanosensor function of TRPV4. Furthermore, the action of TRPV4

agonists 4PDD and GSK1016790A can be counteracted by P2X3 and P2X2/3 antagonists

TNT-ATP, A317491 and PPADS, further validating the upstream involvement of TRPV4

in this signaling pathway [26, 69].

Further validation of the potential of TRPV4 antagonism for the treatment of

bladder disorders involving reduced bladder function and increased micturition frequency

was published recently [47]. These researchers observed that TRPV4 deletion

significantly reduced cystitis-induced bladder dysfunction. Furthermore, similar results

were obtained using a novel TRPV4 antagonist, HC-067047[47]. The lack of effect of

HC-067047 in TRPV4 KO animals strongly suggests its efficacy was due to TRPV4

antagonism.

Expressed in the bladder urothelium and smooth muscle cells, TRPV4 is a major

component of the mechanosensor monitoring bladder distension and its activity controls,

8

in part, voiding of the bladder [26, 27, 43]. Evidence is accumulating that TRPV4

antagonism is correlating with decreased micturition frequency and increased voided

volumes. Furthermore, due to the expression of TRPV4 in the urethra and ureters, it has

been suggested that it may be involved in the urethra-to-bladder reflex facilitating

complete voiding [27]. Overall, these data paint a promising profile for the treatment of

overactive bladder syndrome, cystitis and prostate hyperplasia through antagonism of

TRPV4.

Acute lung injury

Acute lung injury and acute respiratory distress syndrome affect 200,000 patients

every year in the United States, with limited treatment options leading to a high mortality

rate [73, 74]. These ailments are characterized by disruption of the alveolar septal barrier,

hypoxemia and patchy alveolar flooding [75]. Mechanical ventilation is responsible for a

significant part of these injuries [76]. Previous studies have revealed the increase in lung

vasculature permeability to be initiated by intracellular calcium influx, which itself can

be prompted by high airway pressure (mechanical stress) [77].

Hamanaka et al hypothesized that TRPV4 may be the mechanosensor at the origin

of this injury process [78]. The permeability increase linked to high airway pressure was

indeed observed to be abolished in TRPV4 KO mice. This result could be replicated in

TRPV4 WT animals by using ruthenium red, a broad ion channel inhibitor [52], and

inhibitors of arachidonic acid production (methanandamide) and its metabolism to EETs

(miconazole) [20, 78]. Similarly, after confirming the expression of TRPV4 in situ,

Alvarez and coworkers monitored lung endothelium permeability in response to TRPV4

9

agonists 4PDD and 5,6-EET in TRPV4 WT and null mice [79]. Notably, the increase in

permeability observed in response to the application of the agonists in TRPV4 WT mice

was abolished in TRPV4 KO animals. Additionally, application of ruthenium red to WT

lungs stimulated by channel agonists produced similar results, further confirming the

involvement of TRPV4 [79]. As macrophage depletion can attenuate ventilator-induced

lung injury, subsequent work focused on the role of alveolar macrophages, believed to be

key early actors in the injury process [80, 81]. Instillation of TRPV4+/+ macrophages in

TRPV4-/- mice restored the permeability increase observed in response to high airway

pressure, confirming the involvement of both macrophages and TRPV4.

In summary, while early reports linked TRPV4 to high airway pressure-mediated

lung vasculature permeability, recent studies provided additional mechanistic information

on the pathways involved [80, 82]. Given the role of this ion channel as an early enabler

of injury, TRPV4 antagonism holds promise for the treatment of acute lung injury,

especially that caused by mechanical ventilation.

TRPV4 agonists and antagonists

TRPV4 agonists

Phorbol esters as TRPV4 agonists

The phorbol ester, 4α phorbol 12,13-didecanoate (4 αPDD) 1, was the first TRPV4

agonist to be described in the literature [83]. Unlike many phorbol esters, 4αPDD does

not serve as an activator of protein kinase C (PKC) at concentrations up to 25 μM [83,

84].

10

Analogues related to 4αPDD have also been evaluated as TRPV4 agonists and

have revealed important facets of the binding of phorbol esters to TRPV4 (Figure 1) [85].

For example, 4αPDD has an EC50 of 0.37 μM (± 0.08 μM) at TRPV4. Shortening the

ester chain length from 10 carbons found in 4αPDD, to 6 carbons, giving dihexanoate

ester 2, resulted in an increase of EC50 to 0.07 μM (± 0.01 μM). Interestingly, other

lengths of ester side-chain resulted in a dramatic decrease in TRPV4 activity. During

these structure-activity studies, it was also found that an hydroxyl group at the 5,7-ring

juction of the phorbol skeleton was very important for TRPV4 activity, since its removal

to give compound 3, abolished agonism of TRPV4.

Phorbol esters are reported to interact with the TRPV4 ion-channel directly [5,

83-85], and in contrast to the binding of phorbols to PKC, do not use a “typical” cysteine-

rich phorbol binding site [85].

4αPDD has been a very important ligand for investigation of the role of TRPV4,

being used to study effects upon pain [53, 86], bladder function [26], and control of blood

pressure [87, 88], amongst others.

Figure 1. Phorbol esters as agonists of TRPV4.

11

GSK1016790A

GSK1016790A 4 (from GlaxoSmithKline) was recently disclosed as a very potent

TRPV4 agonist (EC50 hTRPV4 = 5 nM) [43]. Significantly, GSK1016790A evoked a

much greater current density at TRPV4 when compared with 4αPDD, indicating greater

relative efficacy. The starting point for the discovery of GSK1016790A was the known

Cathepsin K inhibitor 5, which was found to be a sub-micromolar agonist of TRPV4

(Figure 2). Subsequent optimization of compound 5 gave-rise to GSK1016790A, and a

whole series of structurally-related agonists [42, 43, 89, 90].

GSK101670A is reported to be selective against TRPV1, but may interact with

other receptors, as exposure of non-TRPV4 expressing HEK293 cells to GSK1016790A

results in calcium uptake at low concentrations (50-100 nM) [42, 43]. GSK101670A has

been utilized for a number of in vivo investigations. For example, it induced bladder

hyperactivity in mice [43]. However, work with GSK1016790A has been somewhat

limited, as a result of lethal TRPV4-mediated circulatory collapse in mice, rats and dogs,

upon exposure to an intravenous injection of this TRPV4 agonist [42]. Naturally, this

result indicates that the use of systemic TRPV4 agonists in human clinical trials may be

limited.

Figure 2. GSK101670A as a TRPV4 agonist

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RN-1747

RN-1747 6 was identified as a sub-micromolar TRPV4 agonist during a screen of

commercial arylsulfonamides as TRPV4 ligands (EC50 = 0.77 μM) [91]. RN-1747 is

relatively selective for TRPV4, although it serves as a TRPM8 antagonist at higher

concentrations (IC50 = 4 μM).

Figure 3. RN-1747 as a TRPV4 agonist

Endogenous agonists of TRPV4

Lipid metabolites of the arachidonic acid pathway have been shown to serve as

endogenous agonists of TRPV4 [5, 19, 20, 83]. For example, 5,6-epoxyeicosatrienoic

acid 7 (5,6-EET) and 8,9-epoxyeicosatrienoic acid 8 (8,9-EET) were identified by Nilius

13

and co-workers as TRPV4 agonists, with an EC50 of 0.15 μM versus hTRPV4. Whether

they interact directly with the ion channel is still uncertain however [4].

Figure 4. Lipid metabolites of the arachidonic acid serving as TRPV4 agonists

TRPV4 Antagonists

Ruthenium red

Ruthenium red 9, a metal-based dye acting as a pore blocker, was one of the first potent

TRPV4 antagonists to be identified [15, 83]. However, ruthenium red is an unselective

ligand as activity has been reported with more than 20 other ion channels and biological

targets [52]. Nonetheless, ruthenium red has been used extensively as a probe of TRPV4

activity in the in vivo setting [86, 88, 92].

Figure 5. Ruthenium red, a TRPV4 antagonist

HC-067047

In 2010, Hydra Biosciences disclosed the structure of HC-067047 10, as a potent TRPV4

antagonist (Figure 6) [47]. This compound is a potent inhibitor of the human, mouse, and

rat orthologs of TRPV4 (IC50 of 48 nM, 17 nM and 133 nM, respectively). HC-067047

14

also inhibits TRPM8 and the hERG potassium channel, with an approximate ten-fold

window of selectivity at TRPV4 over these other ion channels. The activity at the hERG

potassium channel may limit the use of HC-067047 to the preclinical setting, as inhibition

of hERG has been associated with QTc prolongation, a potentially severe cardiovascular

side effect [93]. In vivo, HC-067047 was shown to improve bladder function in rodent

models of cystitis, induced by cyclophosphamide [47]. Other in vivo measurements

indicated that HC-067047 did not affect core body temperature, thermal selection

behaviour, water intake, heart rate locomotion, or motor coordination. Thus, HC-067047

may serve as an important tool compound to probe the functions of TRPV4 in vivo.

Figure 6. HC-067047, a TRPV4 antagonist active in rodent models of cystitis

GSK-205 and other antagonists from GlaxoSmithkline (GSK)

Extending their work beyond TRPV4 agonists, GSK have also reported on the design and

synthesis of TRPV4 antagonists. For example, GSK have reported on the use of an

aminothiazole ligand 11, named GSK-205, as a sub-micromolar selective inhibitor of

TRPV4 in porcine chondrocytes (IC50 = 0.6 μM) [94]. Additionally, GSK have also

detailed a number of different ligands as TRPV4 antagonists. Representative structures of

some compounds described in recent patent applications are shown in Figure 7 [95-98].

15

Figure 7. GSK-205 and other representative TRPV4 antagonists from GlaxoSmithKline

RN-1734 and RN-9893

RN-1734 13 was identified in the same focused arylsulfonamide screen as TRPV4

agonist RN-1747 described earlier [91]. RN-1734 inhibits TRPV4 in the micromolar

range when 4αPDD, or hypotonicity, is used as the activating stimuli (IC50 hTRPV4 ca.

2.3 μM). A somewhat more potent TRPV4 antagonist, RN-9893, has also been reported,

but the structure has not been disclosed at the time of writing this mini review [99].

Figure 8. RN-1734 as a TRPV4 antagonist

Vanilloids and TRPV1 ligands as TRPV4 antagonists

It comes as no surprise that certain vanilloids and literature TRPV1 antagonists may have

activity at TRPV4. For example, antagonist activity at TRPV4 has been described for

vanilloid 14 and the quintesential TRPV1 antagonist, capsazepine 15 [91, 100]. However,

16

in the case of capsazepine, the IC50 for inhibition of TRPV4 (15 μM) is significantly

higher than its IC50 against TRPV1 (5-50 fold, depending on the assay) [91, 101].

Figure 9. Vanilloid derivative and the well-known TRPV1 antagonist, Capsazepine as

TRPV4 antagonists

Conclusion

With its wide expression pattern and multiple activating modalities, TRPV4 plays

a variety of physiological roles, including several related to disease states. Potent,

bioavailable and relatively selective TRPV4 modulators such as agonist GSK1016790A 4

and antagonist HC-067047 10 are now available to help validate TRPV4 modulation

against specific diseases. Of special interest are the potential therapeutic benefits of

TRPV4 antagonism for the treatment of inflammatory and neuropathic pain, bladder and

urinary disorders as well as ventilator-induced lung injury, all areas with significant

unmet medical needs. While TRPV4-directed compounds are often active against other

TRP channels, selectivity appears to be an achievable goal. On the other hand, the

multiplicity of physiological roles carried out by this ion channel suggests that careful

evaluation of potential on-target toxicity will be necessary in any drug discovery project

targeting TRPV4.

17

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