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Arpad Szallasi and Tamás Bíró (eds.), TRP Channels in Drug Discovery: Volume I, Methods in Pharmacology and Toxicology, DOI 10.1007/978-1-62703-077-9_13, © Springer Science+Business Media, LLC 2012

Chapter 13

TRPV4 and Drug Discovery

Fabien Vincent and Matthew A. J. Duncton

Abstract

Transient receptor potential vanilloid 4 (TRPV4) was fi rst identi fi ed 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 pro fi le, 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 has 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 signi fi cant unmet medical needs such as in fl ammatory and neuropathic pain, bladder dysfunctions, 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.

Key words: TRPV4 , Agonist , Antagonist , Disease , Drug , Indication , Inhibitor , Activator , Pain , Bladder , Lung , Injury , In fl ammation , Neuropathy , Chemistry

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 nonselective cation permeable channels (P Ca /P Na £ 10) highly sensitive to temperature changes (TRPV1-4) and Ca 2+ selective channels (P Ca /P Na > 100) insensitive to temperature variations (TRPV5-6) ( 3– 5 ) . TRPV1, the canonical member of this family, was fi rst identi fi ed as the receptor of the active ingredient of chili peppers, capsaicin ( 6 ) . Additional studies uncovered other activating modalities such as noxiously high temperatures (>42°C), acidity

1. Introduction

258 F. Vincent and M.A.J. Duncton

(<pH 5), 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 sen-sor, integrating a variety of activating signals to produce a resulting cellular output ( 8 ) .

TRPV4 is closely related to the TRPV1 receptor, sharing 40.9% of its sequence. The human gene is located on chromosome 12q23-q24.1, with fi ve proposed splice variants produced from 12 exons ( 9, 10 ) . The 871 amino acids of full length hTRPV4 de fi ne 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 fi rst ankyrin domain, and putative calmodulin domains near the C terminus ( 4, 5, 9 ) . As with TRPV1, TRPV4 is believed to form homotetramers 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 identi fi ed, including warm temperatures (>27°C) ( 15 ) , phospho-rylation 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 b 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 chapter—the reader is referred to several recent reviews ( 4, 9, 41 ) —while some areas of interest for drug discovery will be expanded upon. Brie fl y, functional studies investigating TRPV4 functions were conducted using a range of tools including small molecule agonists and antag-onists, antisense oligodeoxynucleotides, KO mice, and speci fi c 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

25913 TRPV4 and Drug Discovery

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.

TRPV4 is expressed in peripheral nociceptive neurons (DRG and trigeminal (TG) neurons), with data suggesting its transport to periph-eral nerve endings ( 32, 33 ) . As TRPV4 was originally identi fi ed as an osmosensor, early work using TRPV4 KO investigated whether this channel was also involved in mechanosensation. These studies pro-vided the first evidence of TRPV4 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 knockdown 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 in fl ammatory mediator, was originally observed to sensitize this ion channel to both hypo-tonic and hypertonic media, conditions relevant to diabetes and asthma, for example ( 33, 56, 59 ) . This fi nding was later extended through the use of several additional in fl ammatory mediators, leading to the discovery that increased cAMP levels as well as PKA and PKC e activation were necessary for TRPV4-mediated mechan-ical 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 in fl ammation sensitizes TRPV4 through the stimulation of PLC b , PKA, PKC, and PKD ( 60 ) . Several groups have now con fi rmed the link between PAR2 activa-tion 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 hyper-algesia in multiple rodent models of painful peripheral neuropathy ( 34, 55 ) . The wide range of reagents used for nerve injury in these

2. Areas of Interest for Drug Discovery

2.1. In fl ammatory and Neuropathic Pain

260 F. Vincent and M.A.J. Duncton

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 a 2 b 1 integrin in TRPV4-mediated mechan-ical hyperalgesia ( 55 ) . TRPV4 expression levels were observed to increase in a rat model of chronic constriction of the DRG (CCD). TRPV4-speci fi c AS ODN reversed this increase and partially blocked the observed mechanical allodynia while leaving baseline nociception unaffected ( 32 ) . Additionally, the PAR2–TRPV4 rela-tionship was recently found to extend beyond in fl ammatory con-ditions 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 antago-nists may therefore prove therapeutically useful in the treatment of in fl ammatory and neuropathic pain, potentially including visceral pain related to Crohn’s disease and in fl ammatory bowel syndrome.

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 4 a PDD, GSK1016790A, and hypotonicity ( 26, 27, 43 ) . This pharmacological response was, how-ever, reduced (hypotonicity) or absent (4 a PDD 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 ) . These data are consistent with TRPV4 ablation leading to higher bladder fi lling and delayed micturition ( 68 ) . Conversely, use of TRPV4 agonists 4 a PDD 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 selec-tive 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 signi fi cant change in TRPV4 expression was observed in TRPV1 KO animals suggesting these ion channels do not compensate for each other ( 27, 68 ) .

2.2. Bladder Dysfunction

26113 TRPV4 and Drug Discovery

Following the activation of the urothelium mechanosensory system, ATP is released to activate P 2 X 3 and P 2 X 2/3 purinergic channels on sensory afferents. This pressure-initiated ATP release is decreased in TRPV4 null mice, a result consistent with the postulated mechanosensory function of TRPV4. Furthermore, the action of TRPV4 agonists 4 a PDD and GSK1016790A can be counteracted by P 2 X 3 and P 2 X 2/3 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 func-tion and increased micturition frequency was published recently ( 47 ) . These researchers observed that TRPV4 deletion signi fi cantly 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 ef fi cacy 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 antag-onism 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 re fl ex facilitating com-plete voiding ( 27 ) . Overall, these data paint a promising pro fi le for the treatment of overactive bladder syndrome, cystitis, and pros-tate hyperplasia through antagonism of TRPV4.

Acute lung injury and acute respiratory distress syndrome affect 200,000 patients every year in the United States, with limited treat-ment options leading to a high mortality rate ( 73, 74 ) . These ail-ments are characterized by disruption of the alveolar septal barrier, hypoxemia, and patchy alveolar fl ooding ( 75 ) . Mechanical ventila-tion is responsible for a signi fi cant part of these injuries ( 76 ) . Previous studies have revealed the increase in lung vasculature per-meability to be initiated by intracellular calcium in fl ux, which itself can be prompted by high airway pressure (mechanical stress) ( 77 ) .

Hamanaka et al. hypothesized that TRPV4 may be the mecha-nosensor 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 ) (Note: EETs are endogenous agonists of TRPV4- see section 3.1.4). Similarly, after con fi rming the expression of TRPV4 in situ, Alvarez and coworkers monitored lung endothelium permeability

2.3. Acute Lung Injury

262 F. Vincent and M.A.J. Duncton

in response to TRPV4 agonists 4 a PDD 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, applica-tion of ruthenium red to WT lungs stimulated by channel agonists produced similar results, further con fi rming 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, con fi rming the involvement of both macrophages and TRPV4.

In summary, while early reports linked TRPV4 to high airway pressure-mediated lung vasculature permeability, recent studies pro-vided 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.

The phorbol ester, 4 a phorbol 12,13-didecanoate (4 a PDD) 1 , was the fi rst TRPV4 agonist to be described in the literature ( 83 ) . Unlike many phorbol esters, 4 a PDD does not serve as an activator of protein kinase C (PKC) at concentrations up to 25 m M ( 83, 84 ) .

Analogues related to 4 a PDD have also been evaluated as TRPV4 agonists and have revealed important facets of the binding of phorbol esters to TRPV4 (Fig. 1 ) ( 85 ) . For example, 4 a PDD has an EC 50 of 0.37 m M (±0.08 m M) at TRPV4. Shortening the ester chain length from 10 carbons found in 4 a PDD, to 6 carbons, giving dihexanoate ester 2 , resulted in an increase of EC 50 to

3. TRPV4 Agonists and Antagonists

3.1. TRPV4 Agonists

3.1.1. Phorbol Esters as TRPV4 Agonists

Fig. 1. Phorbol esters as agonists of TRPV4.

26313 TRPV4 and Drug Discovery

0.07 m M (±0.01 m 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 a hydroxyl group at the 5,7-ring junction 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 ) .

4 a PDD 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 ) among others.

GSK1016790A 4 (from GlaxoSmithKline) was recently disclosed as a very potent TRPV4 agonist (EC 50 hTRPV4 = 5 nM) ( 43 ) . Signi fi cantly, GSK1016790A evoked a much greater current den-sity at TRPV4 when compared with 4 a PDD, indicating greater relative ef fi cacy. The starting point for the discovery of GSK1016790A was the Cathepsin K inhibitor 5 , which was found to be a submicromolar agonist of TRPV4 (Fig. 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 exam-ple, it induced bladder hyperactivity in mice ( 43 ) . However, work

3.1.2. GSK1016790A

Fig. 2. GSK101670A as a TRPV4 agonist.

264 F. Vincent and M.A.J. Duncton

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.

RN-1747 6 (Fig. 3 ) was identi fi ed as a submicromolar TRPV4 agonist during a screen of commercial arylsulfonamides as TRPV4 ligands (EC 50 = 0.77 m M) ( 91 ) . RN-1747 is relatively selective for TRPV4, although it serves as a TRPM8 antagonist at higher con-centrations (IC 50 = 4 m M).

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-epoxye-icosatrienoic acid 8 (8,9-EET) were identi fi ed by Nilius and coworkers as TRPV4 agonists, with an EC 50 of 0.15 m M versus hTRPV4 (Fig. 4 ). Whether they interact directly with the ion chan-nel is still uncertain, however ( 4 ) .

Ruthenium red 9 (Fig. 5 ), a metal-based dye acting as a pore blocker, was one of the fi rst potent TRPV4 antagonists to be identi fi ed ( 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 ) .

In 2010, Hydra Biosciences disclosed the structure of HC-067047 10 as a potent TRPV4 antagonist (Fig. 6 ) ( 47 ) . This compound is

3.1.3. RN-1747

3.1.4. Endogenous Agonists of TRPV4

3.2. TRPV4 Antagonists

3.2.1. Ruthenium Red

3.2.2. HC-067047

Fig. 3. RN-1747 as a TRPV4 agonist.

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

26513 TRPV4 and Drug Discovery

a potent inhibitor of the human, mouse, and rat ortholog of TRPV4 (IC 50 of 48 nM, 17 nM, and 133 nM, respectively). HC-067047 also inhibits TRPM8 and the hERG potassium channel, with an approximate tenfold 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 behavior, 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.

Extending their work beyond TRPV4 agonists, GlaxoSmithkline (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 submicromolar selective inhibitor of TRPV4 in porcine chondrocytes (IC 50 = 0.6 m M) ( 94 ) . Additionally, GSK have also detailed a num-ber of different ligands as TRPV4 antagonists. Representative structures of some compounds described in recent patent applica-tions are shown in Fig. 7 ( 95– 98 ) .

RN-1734 13 (Fig. 8 ) has been identi fi ed in the same focused arylsulfon-amide screen as TRPV4 agonist RN-1747 described earlier ( 91 ) .

3.2.3. GSK-205 and Other Antagonists from GlaxoSmithkline

3.2.4. RN-1734 and RN-9893

Fig. 5. Ruthenium red, a TRPV4 antagonist.

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

266 F. Vincent and M.A.J. Duncton

RN-1734 inhibits TRPV4 in the micromolar range when 4 a PDD, or hypotonicity, is used as the activating stimuli (IC 50 hTRPV4 ca. 2.3 m 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 chapter ( 99 ) .

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 quintessential TRPV1 antagonist, capsazepine 15 ( 91, 100 ) . However, in the case of capsazepine, the IC 50 for inhibition of TRPV4 (15 m M) is signi fi cantly higher than its IC 50 against TRPV1 (5–50-fold, depending on the assay) ( 91, 101 ) (see Fig. 9 ).

3.2.5. Vanilloids and TRPV1 Ligands as TRPV4 Antagonists

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

Fig. 8. RN-1734 as a TRPV4 antagonist.

Fig. 9. Vanilloid derivative and the well-known TRPV1 antagonist, capsazepine as TRPV4 antagonists.

26713 TRPV4 and Drug Discovery

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 antago-nist HC-067047 10 are now available to help validate TRPV4 modulation against speci fi c diseases. Of special interest are the potential therapeutic bene fi ts of TRPV4 antagonism for the treat-ment of in fl ammatory and neuropathic pain, bladder and urinary disorders, as well as ventilator-induced lung injury, all areas with signi fi cant unmet medical needs. While TRPV4-directed com-pounds are often active against other TRP channels, selectivity appears to be an achievable goal. On the other hand, the multiplic-ity 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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