TRPV4 and Drug Discovery

Fabien Vincent* and Matthew A. J. Duncton*

* Corresponding authors: Fabien Vincent Pfizer, Inc. 558 Eastern Point Rd, Groton, 06340, CT. Tel: (650)-619-0722 fabien_vincent_us@yahoo.com Matthew A. J. Duncton Formerly with Renovis, Inc. (a wholly owned subsidiary of Evotec AG), Two Corporate Drive, South San Francisco, 94080, CA. Tel: (917) 345-3183 mattduncton@yahoo.com

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.

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/PNa10) 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 (> 42C), 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

its modulation by osmolarity [12-14]. Additional stimuli were soon identified, including warm temperatures (>27C) [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].

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 TPRV4s 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

cAMP levels as well as PKA and PKC activation were necessary for TRPV4-mediated mechanical hyperalgesia [54]. Further studies investigated its connection with proteaseactivated 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

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 Crohns 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 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, 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 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, 4 phorbol 12,13-didecanoate (4 PDD) 1, was the first TRPV4 agonist to be described in the literature [83]. Unlike many phorbol esters, 4PDD does

10

not serve as an activator of protein kinase C (PKC) at concentrations up to 25 M [83, 84]. Analogues related to 4PDD 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, 4PDD has an EC50 of 0.37 M ( 0.08 M) at TRPV4. Shortening the ester chain length from 10 carbons found in 4PDD, to 6 carbons, giving dihexanoate ester 2, resulted in an increase of EC50 to 0.07 M ( 0.01 M). 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 cysteinerich phorbol binding site [85]. 4PDD 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.

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O C9H19 H OH O HO OH 4PDD; 1 TRPV4 EC50 = 0.37 M HH O HO O O O C9H19 H C5H11

O O O O C5H11 H OH HH O OH 4PDH; 2 TRPV4 EC50 = 0.07 M C9H19

O O O O C9H19

OH HH OH 3 (deoxygenated version of 4PDD) TRPV4 EC50 = inactive

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 4PDD, 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.

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Figure 2. GSK101670A as a TRPV4 agonist

O S N H O

H N

O N S OO 5; TRPV4 Hit (originally, a Cathepsin K inhibitor) TRPV4 EC50 = 707 nM Cat K IC50 = 2 nM

CN

Medicinal chemistry optimization GSK1016790A 4 TRPV4 Potencies TRPV4 HEK FLIPR = 5.0 nM Human Chondrocytes TRPV4 = 3.0 nM Bovine Chondrocytes TRPV4 = 1.0 nM Mouse Chondrocytes TRPV4 = 18.5 nM Rat Chondrocytes TRPV4 = 10 nM Dog Chondrocytes TRPV4 = 1.0 nM TRPV1 HEK FLIPR = 50 nM

HO S O H N O N N O O N H S O

Cl

Cl

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 M) [91]. RN-1747 is relatively selective for TRPV4, although it serves as a TRPM8 antagonist at higher concentrations (IC50 = 4 M).

Figure 3. RN-1747 as a TRPV4 agonist

NO2 O S N O Ph N

Cl

RN-1747 6 hTRPV4 EC50 = 0.77 M

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

13

acid 7 (5,6-EET) and 8,9-epoxyeicosatrienoic acid 8 (8,9-EET) were identified by Nilius and co-workers as TRPV4 agonists, with an EC50 of 0.15 M 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

O O OH O O OH

5,6-EET 7

8,9-EET 8

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

H2N NH2 H2N NH2 H2N NH2 H2N Ru O Ru O Ru NH2 H2N NH2 H2N NH2 H2N NH2 Ruthenium Red 9

Cl6

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

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

CF3 NH N N O

HC-067047 10 hTRPV4 IC50 = 48 nM rTRPV4 IC50 = 133 nM mTRPV4 IC50 = 17 nM active at 10 mg/kg i.p. in model 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 M) [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].

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

N H N O O N H N O GSK-205 11 IC50 porcine TRPV4 = 0.6 M H O O S N H Cl

N S N NH .HBr

N H

Cl

Representative TRPV4 antagonist 12 (from GSK)

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 4PDD, or hypotonicity, is used as the activating stimuli (IC 50 hTRPV4 ca. 2.3 M). 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

Cl Cl O S N O

HN

RN-1734 13 IC50 hTRPV4 = 2.3 M

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 M) 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
O MeO HO Vanilloid derivative 14 N H C9H19 HO HO N S N H Cl

Capsazepine 15

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.

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References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Damann, N., et al. (2008) TRPs in our senses, Curr. Biol. 18, R880-889. Venkatachalam, K., and Montell, C. (2007) TRP channels, Annu. Rev. Biochem. 76, 387-417. Vennekens, R., et al. (2008) Vanilloid transient receptor potential cation channels: an overview, Curr. Pharm. Des. 14, 18-31. Everaerts, W., et al. (2009) The vallinoid transient receptor potential channel Trpv4: From structure to disease, Prog. Biophys. Mol. Biol., doi:10.1016/j.pbiomolbio.2009.1010.1002. Nilius, B., et al. (2004) TRPV4 calcium entry channel: a paradigm for gating diversity, Am. J. Physiol. Cell Physiol. 286, C195-205. Caterina, M. J., et al. (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389, 816-824. Cortright, D. N., and Szallasi, A. (2004) Biochemical pharmacology of the vanilloid receptor TRPV1. An update, Eur J Biochem 271, 1814-1819. Szallasi, A., et al. (2007) The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept, Nat. Rev. Drug Discov. 6, 357-372. Plant, T. D., and Strotmann, R. (2007) Trpv4, Handb. Exp. Pharmacol., 189-205. Arniges, M., et al. (2006) Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking, The Journal of biological chemistry 281, 1580-1586. Shigematsu, H., et al. (2009) A 3.5-nm structure of rat TRPV4 cation channel revealed by zernike phase-contrast cryo-EM, J. Biol. Chem. Liedtke, W., and Friedman, J. M. (2003) Abnormal osmotic regulation in trpv4-/mice, Proc. Natl. Acad. Sci. USA 100, 13698-13703. Strotmann, R., et al. (2000) OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity, Nat. Cell Biol. 2, 695-702. Liedtke, W., et al. (2000) Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor, Cell 103, 525-535. Guler, A. D., et al. (2002) Heat-evoked activation of the ion channel, TRPV4, J. Neurosci. 22, 6408-6414. Xu, H., et al. (2003) Regulation of a transient receptor potential (TRP) channel by tyrosine phosphorylation. SRC family kinase-dependent tyrosine phosphorylation of TRPV4 on TYR-253 mediates its response to hypotonic stress, J. Biol. Chem. 278, 11520-11527. Fan, H. C., et al. (2009) Activation of the TRPV4 ion channel is enhanced by phosphorylation, J. Biol. Chem. 284, 27884-27891. Peng, H., et al. (2010) Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel, Biochem. Biophys. Res. Commun. 391, 1721-1725. Vriens, J., et al. (2005) Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium, Circ. Res. 97, 908915. Watanabe, H., et al. (2003) Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels, Nature 424, 434-438. 18

17. 18. 19. 20.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

Chung, M. K., et al. (2003) Warm temperatures activate TRPV4 in mouse 308 keratinocytes, J. Biol. Chem. 278, 32037-32046. Fernandez-Fernandez, J. M., et al. (2002) Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line, Am. J. Physiol. Cell Physiol. 283, C1705-1714. Jia, Y., et al. (2004) Functional TRPV4 channels are expressed in human airway smooth muscle cells, Am. J. Physiol. Lung Cell Mol. Physiol. 287, L272-278. Wissenbach, U., et al. (2000) Trp12, a novel Trp related protein from kidney, FEBS Lett. 485, 127-134. Delany, N. S., et al. (2001) Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2, Physiol. Genomics 4, 165-174. Birder, L., et al. (2007) Activation of urothelial transient receptor potential vanilloid 4 by 4alpha-phorbol 12,13-didecanoate contributes to altered bladder reflexes in the rat, J. Pharmacol. Exp. Ther. 323, 227-235. Gevaert, T., et al. (2007) Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding, J. Clin. Invest. 117, 3453-3462. Shen, J., et al. (2006) Functional expression of transient receptor potential vanilloid 4 in the mouse cochlea, Neuroreport 17, 135-139. Mergler, S., et al. (2011) Characterization of transient receptor potential vanilloid channel 4 (TRPV4) in human corneal endothelial cells, Exp Eye Res 93, 710-719. Mizuno, A., et al. (2003) Impaired osmotic sensation in mice lacking TRPV4, Am. J. Physiol. Cell Physiol. 285, C96-101. Shibasaki, K., et al. (2007) Effects of body temperature on neural activity in the hippocampus: regulation of resting membrane potentials by transient receptor potential vanilloid 4, J. Neurosci. 27, 1566-1575. Zhang, Y., et al. (2008) A transient receptor potential vanilloid 4 contributes to mechanical allodynia following chronic compression of dorsal root ganglion in rats, Neurosci. Lett. 432, 222-227. Alessandri-Haber, N., et al. (2003) Hypotonicity induces TRPV4-mediated nociception in rat, Neuron 39, 497-511. Alessandri-Haber, N., et al. (2004) Transient receptor potential vanilloid 4 is essential in chemotherapy-induced neuropathic pain in the rat, J. Neurosci. 24, 4444-4452. Wu, L., et al. (2007) Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells, Am. J. Physiol. Renal Physiol. 293, F16991713. Watanabe, H., et al. (2002) Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells, J. Biol. Chem. 277, 47044-47051. Kohler, R., et al. (2006) Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation, Arterioscler. Thromb. Vasc. Biol. 26, 1495-1502. Kumar, S., et al. (2009) WO2006029209, Chem. Abstr. 144, 286221. Masuyama, R., et al. (2008) TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts, Cell Metab. 8, 257-265.

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40. 41. 42. 43.

44. 45. 46. 47. 48. 49. 50. 51.

52. 53. 54. 55.

Mizoguchi, F., et al. (2008) Transient receptor potential vanilloid 4 deficiency suppresses unloading-induced bone loss, J. Cell Physiol. 216, 47-53. Nilius, B., and Owsianik, G. (2010) Channelopathies converge on TRPV4, Nat. Genet. 42, 98-100. Willette, R. N., et al. (2008) Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2, J. Pharmacol. Exp. Ther. 326, 443-452. Thorneloe, K. S., et al. (2008) N-((1S)-1-{[4-((2S)-2-{[(2,4dichlorophenyl)sulfonyl]amino}-3-hydroxypropa noyl)-1-piperazinyl]carbonyl}3-methylbutyl)-1-benzothiophene-2-carboxamid e (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I, J. Pharmacol. Exp. Ther. 326, 432442. Suzuki, M., et al. (2003) Impaired pressure sensation in mice lacking TRPV4, J. Biol. Chem. 278, 22664-22668. Rock, M. J., et al. (2008) Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia, Nat. Genet. 40, 999-1003. Krakow, D., et al. (2009) Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia, Am. J. Hum. Genet. 84, 307-315. Everaerts, W., et al. (2010) Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis, Proc Natl Acad Sci U S A 107, 19084-19089. Liedtke, W. (2005) TRPV4 as osmosensor: a transgenic approach, Pflugers Arch. 451, 176-180. Lowry, C. A., et al. (2009) That warm fuzzy feeling: brain serotonergic neurons and the regulation of emotion, J. Psychopharmacol. 23, 392-400. Muramatsu, S., et al. (2007) Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation, J. Biol. Chem. 282, 32158-32167. Gao, X., et al. (2003) Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways, J. Biol. Chem. 278, 2712927137. Vincent, F., and Duncton, M. A. (2011) TRPV4 agonists and antagonists, Curr Top Med Chem 11, 2216-2226. Alessandri-Haber, N., et al. (2009) TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization, J. Neurosci. 29, 6217-6228. Alessandri-Haber, N., et al. (2006) A transient receptor potential vanilloid 4dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators, J. Neurosci. 26, 3864-3874. Alessandri-Haber, N., et al. (2008) Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia, J. Neurosci. 28, 1046-1057.

20

56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

66. 67. 68. 69. 70. 71. 72. 73.

Alessandri-Haber, N., et al. (2005) TRPV4 mediates pain-related behavior induced by mild hypertonic stimuli in the presence of inflammatory mediator, Pain 118, 70-79. Chen, X., et al. (2007) Marked attenuation of inflammatory mediator-induced Cfiber sensitization for mechanical and hypotonic stimuli in TRPV4-/- mice, Molecular pain 3, 31. Levine, J. D., and Alessandri-Haber, N. (2007) TRP channels: targets for the relief of pain, Biochim. Biophys. Acta 1772, 989-1003. Vakili, C., et al. (1970) Chemical and osmolar changes of interstitial fluid in acute inflammatory states, Surgical forum 21, 227-228. Grant, A. D., et al. (2007) Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice, J. Physiol. 578, 715-733. Cenac, N., et al. (2008) Transient receptor potential vanilloid-4 has a major role in visceral hypersensitivity symptoms, Gastroenterology 135, 937-946, 946 e931932. Sipe, W. E., et al. (2008) Transient receptor potential vanilloid 4 mediates protease activated receptor 2-induced sensitization of colonic afferent nerves and visceral hyperalgesia, Am J Physiol Gastrointest Liver Physiol 294, G1288-1298. Brierley, S. M., et al. (2008) Selective role for TRPV4 ion channels in visceral sensory pathways, Gastroenterology 134, 2059-2069. Auge, C., et al. (2009) Protease-activated receptor-4 (PAR 4): a role as inhibitor of visceral pain and hypersensitivity, Neurogastroenterol Motil 21, 1189-e1107. Chen, Y., et al. (2011) Proteinase-activated receptor 2 sensitizes transient receptor potential vanilloid 1, transient receptor potential vanilloid 4, and transient receptor potential ankyrin 1 in paclitaxel-induced neuropathic pain, Neuroscience 193, 440-451. Janssen, D. A., et al. (2011) The mechanoreceptor TRPV4 is localized in adherence junctions of the human bladder urothelium: a morphological study, The Journal of urology 186, 1121-1127. Mochizuki, T., et al. (2009) The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures, The Journal of biological chemistry 284, 21257-21264. Skryma, R., et al. (2011) From urgency to frequency: facts and controversies of TRPs in the lower urinary tract, Nature reviews 8, 617-630. Aizawa, N., et al. (2012) Effects of TRPV4 cation channel activation on the primary bladder afferent activities of the rat, Neurourology and urodynamics 31, 148-155. Boudes, M., et al. (2011) [TRPV4, new therapeutic target for urinary problems], Med Sci (Paris) 27, 232-234. Angelico, P., and Testa, R. (2010) TRPV4 as a target for bladder overactivity, F1000 biology reports 2. Everaerts, W., et al. (2008) On the origin of bladder sensing: Tr(i)ps in urology, Neurourology and urodynamics 27, 264-273. Ware, L. B., and Matthay, M. A. (2000) The acute respiratory distress syndrome, The New England journal of medicine 342, 1334-1349.

21

74. 75.

76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89.

Ware, L. B., and Matthay, M. A. (2005) Clinical practice. Acute pulmonary edema, The New England journal of medicine 353, 2788-2796. Bernard, G. R., et al. (1994) The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination, American journal of respiratory and critical care medicine 149, 818-824. Dos Santos, C. C., and Slutsky, A. S. (2000) Invited review: mechanisms of ventilator-induced lung injury: a perspective, J Appl Physiol 89, 1645-1655. Tiruppathi, C., et al. (2002) Role of Ca2+ signaling in the regulation of endothelial permeability, Vascular pharmacology 39, 173-185. Hamanaka, K., et al. (2007) TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs, Am. J. Physiol. Lung Cell. Mol. Physiol. 293, L923-932. Alvarez, D. F., et al. (2006) Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury, Circ. Res. 99, 988-995. Hamanaka, K., et al. (2010) TRPV4 channels augment macrophage activation and ventilator-induced lung injury, Am. J. Physiol. Lung Cell. Mol. Physiol. 299, L353-362. Eyal, F. G., et al. (2007) Reduction in alveolar macrophages attenuates acute ventilator induced lung injury in rats, Intensive care medicine 33, 1212-1218. Jian, M. Y., et al. (2008) High vascular pressure-induced lung injury requires P450 epoxygenase-dependent activation of TRPV4, American journal of respiratory cell and molecular biology 38, 386-392. Watanabe, H., et al. (2002) Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives, J. Biol. Chem. 277, 13569-13577. Vriens, J., et al. (2007) Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4, J. Biol. Chem. 282, 12796-12803. Klausen, T. K., et al. (2009) Modulation of the Transient Receptor Potential Vanilloid Channel TRPV4 by 4alpha-Phorbol Esters: A Structure-Activity Study, J. Med. Chem. 52, 2933-2939. Ding, X. L., et al. (2010) Involvement of TRPV4-NO-cGMP-PKG pathways in the development of thermal hyperalgesia following chronic compression of the dorsal root ganglion in rats, Behav. Brain Res. 208, 194-201. Gao, F., and Wang, D. H. (2010) Hypotension induced by activation of the transient receptor potential vanilloid 4 channels: role of Ca2+-activated K+ channels and sensory nerves, J. Hypertens. 28, 102-110. Gao, F., et al. (2009) Salt intake augments hypotensive effects of transient receptor potential vanilloid 4: functional significance and implication, Hypertension 53, 228-235. Jeong, J. U. R., A.; Chen, X.; Casillas, L.; Lin, M.; Trout, R.; Luengo, J. I.; Lipshutz, D. B.; Votta, B.; Kulkarni, S. G.; Brown, B. S.; Rominger, D.; Yamashita, D. S.; Marquis, R. W. (2009) in 238th ACS National Meeting, pp MEDI-392, Washington, DC, United States.

22

90. 91. 92. 93. 94. 95. 96. 97. 98. 99.

100. 101.

Casillas, L. T., R.; Lin, M.; Rominger, D.; Donovan, B.; Brown, B. S.; Kulkarni, S. G.; Lipshutz, D. B.; Sanjay, K.; Votta, B.; Luengo, J. I.; Marquis, R. (2009) in 238th ACS National Meeting, pp MEDI-451, Washington, DC, United States. Vincent, F., et al. (2009) Identification and characterization of novel TRPV4 modulators, Biochem. Biophys. Res. Commun. 389, 490-494. Tsushima, H., and Mori, M. (2006) Antidipsogenic effects of a TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, injected into the cerebroventricle, Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1736-1741. Pollard, C. E., et al. (2008) Strategies to reduce the risk of drug-induced QT interval prolongation: a pharmaceutical company perspective, Br J Pharmacol 154, 1538-1543. Phan, M. N., et al. (2009) Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes, Arthritis Rheum 60, 30283037. Cheung, M., et al. (2009) WO2009146182, Chem. Abstr. 152, 27389. Cheung, M., et al. (2009) WO2009146177, Chem. Abstr. 152, 27388. Cheung, M., and Eidam, H. S. (2010) WO2010011912, Chem. Abstr. 152, 215581. Cheung, M., et al. (2009) WO2009111680, Chem. Abstr. 151, 359104. Wei, Z.-L. N., M. T.; Acevedo, A.; Zipfel, S.; Defalco, J.; Dourado, M.; Gustafson, A. E.; Steiger, D.; Chi, C.; Yip, V.; Zhang, Q.; Victoria, C. L.; Reubish, D. S.; Spiro, P. A.; Kelly, M. G.; Kincaid, J.; Emerling, D. E.; Duncton, M.; Vincent, F. . (2007) Discovery of a Proof-of-Concept TRPV4 antagonist, in Keystone Symposia on the Transient Receptor Potential Ion Channel Superfamily, Breckenridge, Colorado, USA Tsukamoto, S., and Sawamura, S. (2009) JP2009084290, Chem. Abstr. 150, 438732. Reubish, D., et al. (2009) Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine, Biotechniques 47, iii-ix.

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