trpv4 agonists and antagonists
TRANSCRIPT
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TRPV4 Agonists and Antagonists
Fabien Vincent* and Matthew A. J. Duncton* * Corresponding authors: Fabien Vincent Pfizer, Inc. 558 Eastern Point Rd, Groton, 06340, CT. Tel: (650)-619-0722 [email protected] Matthew A. J. Duncton Formerly with Renovis, Inc. (a wholly-owned subsidiary of Evotec AG), Two Corporate Drive, South San Francisco, 94080, CA [email protected]
2
Abstract
TRPV4 belongs to the TRPV subfamily of Transient Receptor Potential (TRP)
ion channels. This year marks the 10 year anniversary of the discovery of this polymodal
ion channel which is activated by a variety of stimuli including warm temperatures,
hypotonicity and endogenous lipids. Coupled with a widespread tissue distribution, this
activation profile has resulted in a large number of disparate physiological functions for
TRPV4. These range from temperature monitoring in skin keratinocytes to osmolarity
sensing in kidneys, sheer stress detection in blood vessels and osteoclast differentiation
control in bone. As knowledge of its physiological roles has expanded, interest in
targeting TRPV4 modulation for therapeutic purposes has arisen and is now focused on
several areas. First, as with related TRP channels TRPV1, TRPV3, TRPM8 and TRPA1,
TRPV4 antagonism is being considered for inflammatory and neuropathic pain treatment.
Recent work conducted using KO mice and agonists 4PDD and GSK1016790A
suggests bladder dysfunctions may also be targeted. Additionally, ventilator-induced lung
injury has emerged as another potential indication for TRPV4 antagonists. Herein we
review the known small molecule modulators of TRPV4 and relate progress made in
identifying potent, selective and bioavailable agonists and antagonists to interrogate this
ion channel in vivo.
Keywords: 4PDD, activator, blocker, GSK1016790A, inhibitor, phorbol ester, RN-
1747, RN-1734, ruthenium red.
3
Introduction
The TRP ion channel superfamily comprises 28 members and is divided between
the TRPA, TRPC, TRPM, TRPN, TRPP and TRPV families.[1,2] The TRPV (vanilloid)
channel family is composed of TRPV1 to TRPV6, and can be further subdivided into two
groups with TRPV1-4 being non selective cation permeable channels highly sensitive to
temperature changes and TRPV5-6 being Ca2+ selective channels insensitive to
temperature variations.[3] The canonical member of this family, TRPV1, was identified
as the receptor of capsaicin, the main active ingredient of the chili pepper.[4] Further
work revealed it to be sensitive to other activating modalities as well including noxiously
high temperatures, acidity (low pH), and lipids such as anandamide.[5,6] Overall, TRPV1
was determined to function as a polymodal sensor, integrating a variety of inputs to
produce a resulting cellular signal.
A close relative of the TRPV1 receptor with 40.9% sequence identity, TRPV4
consists of 871 amino acids encompassing three ankyrin repeats near the NH2 terminus
and six transmembrane domains with the pore region being located between TM5 and
TM6.[7,8] Recent work using detergent-solubilized rat TRPV4 and cryo-electron
microscopy suggests the channel is a tetramer.[9,10] Species differences appear limited
with roughly 95% identity being observed for the human sequence as compared with its
rat and mouse counterparts.[7,8,10] Although TRPV4 was originally characterized as an
osmosensor,[11-13] further work revealed it to be activated or sensitized by multiple
other stimuli including warm temperatures (>27°C),[14] phosphorylation by Src, PKC
and PKA,[15-17] and endogenous lipids.[10,18,19]
4
Expression studies have documented a wide tissue distribution for TRPV4 with its
presence being detected in kidney, lung, brain, dorsal root ganglia (DRG), bladder, skin,
vascular endothelium, liver, testis, fat, cochlea and heart.[12,13,20-27] Correspondingly,
TRPV4 is expressed in a number of cell types, both excitable and non-excitable,
including peripheral, hippocampal and subfornical organ neurons, renal epithelial cells,
airway smooth muscle cells, chondrocytes, osteoclasts, epithelial cells of the aorta and
others.[3,11,22,28-38]
Although TRPV4 was discovered only ten years ago, functional studies conducted
with small molecule agonists, antisense oligodeoxynucleotides, KO mice, or based on
specific genetic mutations linking it to rare diseases have exposed a diverse range of roles
for this channel.[11,14,25,28,31,39-43] Several recent reviews cover the biological
functions of TRPV4 in detail.[8,10,44] Briefly, TRPV4 was shown to be involved in
osmolarity sensing and regulation in the CNS,[11-13,28,45] thermosensation and
possibly thermoregulation,[14,20,46] bone formation and remodeling,[36,38,42,43,47]
and mechanosensation in the vascular endothelium.[35,48] Additionally, brachyolmia,
scapuloperoneal spinal muscular atrophy and Charcot-Marie-Tooth disease type 2C were
found to have their origin in specific (activating) TRPV4 mutations.[42-44,49-51]
Following the identification of TRPV4 in DRG and trigeminal (TG) neurons, its role in
nociception has been explored in depth.[30,31] Both neuropathic and inflammatory
conditions were observed to sensitize or activate this receptor, leading to mechanical and
thermal hyperalgesia.[30-32,41,52-56] More recently, the involvement of TRPV4 in
bladder function was investigated by several research groups.[25,26,40,57] Expressed in
the bladder urothelium and smooth muscle cells, TRPV4 was hypothesized to be part of
5
the mechanosensor monitoring bladder distension and controlling, in part, its
voiding.[25,26,40] Finally, TRPV4 has also been implicated in the acute pulmonary
vascular permeability increase observed in response to the inflation pressure caused by
mechanical ventilation.[58-60]
TRPV4 agonists
4PDD
The first small molecule agonist of TRPV4 channels was discovered amongst
phorbol esters.[61] Much as phorbol ester RTX had been found to be a TRPV1
agonist,[62] 4-phorbol 12,13-didecanoate (4PDD) 1 potently activated both human
and mouse TRPV4 (Table 1). Importantly, and unlike phorbol 12-myristate 13-acetate
(PMA), 4PDD is not an activator of PKC at low concentrations (ED50 > 25 M), and
therefore interacts with the ion channel directly.[7,61,63] As it does not activate other
members of the TRPV family, 4PDD is a selective agonist of TRPV4.[7] It has been
administered in vivo using a variety of approaches to investigate the role of TRPV4 in
pain (local or intrathecal injections),[64,65] osmosensing in the CNS
(intracerebroventricular injection),[66] bladder function (intravesicular application),[25]
and blood pressure control (intravenous injection).[67,68]
Table 1. Data summary for representative TRPV4 agonists.
Agonist
Compound number
Potency (EC50, M)
Species
References
6
4PDD 5,6-EET Bisandrographolide RN-1747 GSK-1016790A
1 12 14 15 25
0.16, 0.92 4.4 0.16, 0.37, 0.93 0.15 0.79-0.95 0.77 4.0 4.1 0.005; 0.003a 0.001a
0.0185a
0.010a
0.001a
human rat mouse human mouse human mouse rat human bovine mouse rat dog
[61,69] [69] [61,69,70] [19] [71] [69] [69] [69] [39] [39] [39] [39] [39]
a Chondrocytes from the indicated species were used for this determination.
SAR of analogues related to 4PDD
The investigation of structure-activity relationships around 4PDD derivatives,
and a detailed investigation of the mechanistic aspects of TRPV4 activation with phorbol
esters has been examined by Nilius, and a collaboration between the research groups of
Nilius and Appendino.[61,70] The results from these studies are illuminating to the
medicinal chemist. For instance, in contrast to its activity at protein kinase C (PKC) and
other phorbol ester receptors, it is unlikely that 4PDD 1 binds to TRPV4 using a
cysteine-rich phorbol-binding site, since a “typical” cysteine-rich phorbol binding-site is
not present in TRPV4.[61] Detailed studies with multiple phorbol esters, examining the
esterification pattern on ring C and the nature of the A,B-rings, in combination with site-
directed mutagenesis studies, has provided information on the nature of the binding of
these compounds to TRPV4, and the preferences of the phorbol binding site.[70] For
example, examination of a series of 12,13-diesters against mouse TRPV4 shows two
7
maxima of activity with variation of chain length (Table 2). Thus, the most potent
compounds are those with a 6-carbon chain length (4PDH 4; EC50 = 70 nM; entry 3)
and a 10-carbon chain (4PDD 1; EC50 = 0.37 M; entry 6). Congeners possessing a 2-,
4-, 8-, or 9-carbon tail are essentially inactive against TRPV4. The 12,13-dimyristate 7
(entry 7), with a 14-carbon tail, shows some TRPV4 agonist activity, although the EC50 is
somewhat higher when compared to the 4PDD parent. Deletion of the 12-ester group in
4PDD to give compound 8 results in a compound with diminished relative efficacy to
4PDD, although the EC50 value of 0.45 M was similar to that of 4PDD itself (entry
8). The authors also examined the activity of 4PDH and 4PDD, the most potent
compounds identified from their studies, against mutated versions of TRPV4. This led to
the conclusion that the acyl moieties of phorbol esters serve to position the diterpenoid
core for binding with TRPV4, rather than directly interacting with the binding site.
Table 2. SAR for representative phorbol ester TRPV4 agonists
O
H
HO
OH
OH
H
O
H
O
O
R2
R1
Entry Compound Abbreviation R1 R2 EC50 TRPV4 (M)
1 2 _ COCH3 (Ac) COCH3 (Ac) >50
2 3 _ n-COC3H7 n-COC3H7 22.47 ± 4.04
3 4 4PDH n-COC5H11 n-COC5H11 0.07 ± 0.01
8
4 5 _ n-COC7H15 n-COC7H15 >50
5 6 _ n-COC8H17 n-COC8H17 >50
6 1 4PDD n-COC9H19 n-COC9H19 0.37 ± 0.08
7 7 _ n-COC13H27 n-COC13H27 2.83 ± 0.62
8 8 _ H n-COC9H19 0.45 ± 0.08
Changes to the A,B ring have also been investigated (Figure 1). Interestingly,
4PDD 9, an epimer of 4PDD, is a potent TRPV4 agonist with a similar EC50 value to
4PDD itself.[61] Removing the 4-hydroxyl group in 4PDD resulted in compound 10
which was unable to activate TRPV4. No reports on the ability of this deoxygenated
compound to block the stimulation of TRPV4 with 4PDD were reported, so it is
difficult to ascertain whether this lack of activity was due to a complete abolition in the
binding of the deoxygenated compound 10 to TRPV4, or an inability to evoke an agonist
response after binding. In principle, it is therefore possible that compound 10, the
deoxygenated analogue of 4PDD, might be a TRPV4 antagonist. Finally, one of the
most striking studies performed by Nilius, Appendino and co-workers was in examining
the TRPV4 agonist activity with the lumiphorbol derivative 4LPDD 11, a cage-like (2-
2) photocyclized product of 4PDD. Although studies indicated some similarities in
binding to 4PDD, there were substantial differences in the currrent elicited by the two
agonists. More significantly, and in contrast to both 4PDD 1 and 4PDH 4, 4LPDD
11 maintained the same current densities irrespective of the presence of Ca2+ in the
recording solutions. This, together with other observations indicated the importance of
the A,B-ring for functional agonist TRPV4 activity. The differences in the characteristics
9
of the currents elicited by 4LPDD and 4PDD led the authors to conclude that although
these two ligands bind TRPV4, apparently using the same binding site, their diterpenoid
cores interact with the receptor in different ways.
Figure 1. SAR with phorbol ester and lumiphorbol ester TRPV4 agonists
O
H
HO
OH
OH
H
O
H
O
O
C9H19
O
C9H19
O
H
HO
OH
OH
H
O
H
O
O
C9H19
O
C9H19
O
H
OH
OH
H
O
H
O
O
C9H19
O
C9H19
OH
H
O
H
O
O
C9H19
O
C9H19
O
OH
HHO
4PDD; 6TRPV4 EC50 = 0.37 M
4PDD; 9TRPV4 EC50 similar to 4PDD [58]
10TRPV4 EC50 = inactive
4LPDD; 11TRPV4 EC50 = 0.18 M
5,6-EET and 8,9-EET
As TRPV1 is directly gated by anandamide and other lipids,[72] the search for
endogenous modulators of TRPV4 logically started with the testing of lipids.[7,19] After
observing that anandamide and its metabolite arachidonic acid could activate TRPV4
indirectly,[19,61] Nilius and co-workers explored the downstream metabolism pathways
of arachidonic acid to isolate the eicosanoids directly responsible for this
activation.[18,19] Through the judicious use of inhibitors of the COX, LOX and P450
pathways, they uncovered epoxyeicosatrienoic acids 5,6-EET 12 and 8,9-EET 13 as the
apparent active species (Figure 2). Further studies indicated CYP2C9 to be the key
enzyme responsible for the synthesis of these two agonists.[18] Whether these molecules
directly interact with the channel is still unclear at this time however.[10] This pathway
nonetheless appears to play a key role in the activation of TRPV4 by hypotonicity, as cell
10
swelling induces PLA2 activation and the subsequent release of arachidonic acid from the
membrane.[18,19]
Figure 2. TRPV4 agonists 5,6-EET, 8,9-EET, Bisandrographolide A and RN-1747.
O
O
OH
O
H
OH
OHBisandrographolide A (BAA) 14
O
OH
O
5,6-EET 12
OH
O
8,9-EET 13
O
S
NO2
ClO
ON N
Ph
RN-1747 15
OH
OH
Bisandrographolide A
In a screen of Chinese medicinal herbs against mouse TRPV4, an extract of
Andrographis paniculata was observed to activate the channel.[71] Bisandrographolide A
14 (Figure 2) was subsequently isolated as the active ingredient and was determined to
possess sub-micromolar potency (Table 1). Testing conducted against TRPV1-3 indicated
Bisandrographolide A was selective for TRPV4.
RN-1747
RN-1747 15 (Figure 2) was originally discovered in a focused screen of
commercial ortho- and para-substituted aryl sulfonamides.[69] RN-1747 was shown to
activate human, rat and mouse TRPV4 in the sub-micromolar to low micromolar range
(Table 1). Its efficacy was similar to 4PDD against hTRPV4, but somewhat lower than
that of 4PDD against rTRPV4 and mTRPV4. In a small TRP selectivity panel, RN-1747
11
was selective with respect to TRPV3, induced low-level activation (25% of capsaicin
Emax) of hTRPV1 at 100 M, and antagonized TRPM8 (IC50 = 4 M).
GSK1016790A and patent applications from GlaxoSmithKline (GSK):
Researchers from GSK have published numerous patent applications detailing
small molecules as TRPV4 agonists.[36,73-79] As disclosed at a recent American
Chemical Society National Meeting,[80,81] compound 16 – a known inhibitor of
Cathepsin K – served as an initial hit (Figure 3). Fixing the stereochemistry to (R)- at the
4-position of the azepan-3-one ring resulted in improved selectivity over Cathepsin K
when compared to the (S)-counterpart (see compounds 17 and 18). Removing the
carbonyl group from the 7-membered ring and changing the benzothiophene moiety to an
N-methyl indole eliminated Cathepsin K activity (compound 19). Additional structure-
activity relationships for this class of compound are also detailed in Figure 2 (a Figure
from reference [72] was used to construct this SAR illustration). However, poor
ADME/PK profiles and a narrow structure-activity profile for this class of compound
prompted a search for alternatives to the azepanone/azepine linker.
Figure 3. Representative TRPV4 agonists and SAR information from GSK
12
N
OHN
ONH
O
SO O CN
S
16TRPV4 EC50 (HAC assay) = 707 nM
Cat K IC50 = 2 nM
N
OHN
ONH
O
SO O CN
S
17TRPV4 EC50 (HAC assay) = 110 nM
Cat K IC50 = 180 nM
N
HN
ONH
O
SO O CN
N
18TRPV4 EC50 (HAC assay) = 280 nM
Cat K IC50 > 10000 nM
N
HN
ONH
O
SO O CN
N ortho-CN only: All other inactive
Sulfonamide replacements such as amide or alkyl were inactive
(R)-stereochemistry preferred
>15 amino acids evaluated(L)-Leu optimal
Tight SAR - few otherheterocycles tolerated(e.g. benzothiophenecan replace indole)
N-Methylation of amidesnot well tolerated
19
Futher studies showed that the azepanone/azepine group could be replaced by a
piperazine, morpholine, pyyrolidinol and an aliphatic diamine linker, amongst others.[73-
77,79] Some results with aliphatic diamine compounds serve to illustrate how the
ADME/PK properties of the compounds could be improved.[80] For instance, in a series
of substituted 1,4-diaminobutane analogues, a 2-chloro-4-fluorosulfonamide moiety was
found to be optimal for rat PK, giving dramatic improvements in bioavailability when
compared to a 2,4-dichlorosulfonamide congener (Figure 4).
Figure 4. SAR and rat PK characteristics with diaminobutane TRPV4 agonists
21TRPV4 EC50 FLIPR = 7 nM
TRPV4 EC50 (HAC assay) = 210 nMRat PK
CL = 8.8 mL/min/KgVd = 0.4 L/Kg
%F = 88%
HN
ONH
O
SNH
SO O Cl
Cl
HN
ONH
O
SNH
SO O Cl
F20
TRPV4 EC50 FLIPR = 7 nMTRPV4 EC50 (HAC assay) = 170 nM
Rat PKCL = 11.1 mL/min/Kg
Vd = 0.3 L/Kg%F = 20%
13
Results similar to those illustrated above were also obtained for substituted 1,3-
diaminopropane counterparts (Figure 5).[80] Additionally, introduction of a hydroxyl
group in the 1,3-diaminopropane chain resulted in a key compound (24) that maintained
an excellent balance of functional activity and rat PK characteristics. Interestingly, the 2-
(R) isomer of compound 24 was found to be about 20-50 fold more active than the
corresponding 2-(S) isomer.[80]
Figure 5. SAR and rat PK characteristics with diaminopropane TRPV4 agonists
23TRPV4 EC50 FLIPR = 59 nM
TRPV4 EC50 (HAC assay) = 120 nMRat PK
CL = 2.2 mL/min/KgVd = 0.3 L/Kg
%F = 70%
HN
ONH
O
S
HN
S
22TRPV4 EC50 FLIPR = 20 nM
TRPV4 EC50 (HAC assay) = 130 nMRat PK
CL = 7.6 mL/min/KgVd = 0.4 L/Kg
%F = 34%
OO Cl
ClHN
ONH
O
S
HN
SOO Cl
F
24TRPV4 EC50 FLIPR = 20 nM
TRPV4 EC50 (HAC assay) = 20 nMRat PK
CL = 2.6 mL/min/KgVd = 0.3 L/Kg
%F = 41%
HN
ONH
O
S
HN
SOO Cl
FOH
Finally, GSK has published details on the in vitro and in vivo profile of
GSK1016790A 25, a piperazine-linked TRPV4 agonist (Figure 6).[39,40,81,82] Of note,
was the relative efficacy of GSK1016790A when compared to 4PDD – the TRPV4
current density being much greater for GSK1016790A than for 4PDD (see reference
[40] - Figure 2). Additionally, it should be noted that GSK1016790A is also capable of
activating TRPV1 at low concentrations (EC50 = 50 nM), even though it displays 10-fold
greater potency for TRPV4 (EC50 = 5 nM).[40] When infused into the bladders of mice,
GSK1016790A induced bladder overactivity, an effect that was absent when the
compound was infused into the bladders of TRPV4 knockout mice. This led the authors
to conclude that TRPV4 demonstrated a functional role as regulators of urinary bladder
activity.[40] However, when GSK1016790A was administered intravenously to mouse,
14
rat or dog, a dose-dependent reduction in blood pressure, followed by circulatory collapse
and death was observed.[39] Again, this effect was absent in TRPV4 knockout mice,
indicating it was mediated by the TRPV4 receptor. Mechanistic investigations conducted
in rat and dog pointed to a profound reduction in cardiac output, most likely a result of
disruption to the microvascular permeability barrier, caused by alteration to endothelial
morphology and integrity.[39]
Figure 6. Structure and in vitro profile of GSK1016790A
HN
OS
O
NN
ONH
SO O
HO
Cl Cl
25 GSK1016790A
TRPV4 PotenciesTRPV4 HEK FLIPR = 5.0 nM
Human Chondrocytes TRPV4 = 3.0 nMBovine Chondrocytes TRPV4 = 1.0 nMMouse Chondrocytes TRPV4 = 18.5 nM
Rat Chondrocytes TRPV4 = 10 nMDog Chondrocytes TRPV4 = 1.0 nM
TRPV1 HEK FLIPR = 50 nM(potencies copied from reference 40)
TRPV4 antagonists
Ruthenium red
While gadolinium (Gd3+) and lanthanum (La3+) had already been reported to
partly inhibit TRPV4 activity at 100 M,[12] metal-based dye ruthenium red 26
(structure not shown) was the first potent antagonist of the ion channel to be discovered
(Table 3).[61] Already known as a TRPV1 pore blocker, it was observed to fully
antagonize mouse and human TRPV4 at 1 M in a voltage-dependent manner. Further
characterization revealed ruthenium red to also inhibit rat TRPV4 activity.[14] Selectivity
is a significant issue with ruthenium red as it is known to interact with a number of other
proteins, including mammalian ion channels (CatSper1, TASK, RyR1, RyR2, RyR3,
15
TRPM6, TRPM8, TRPV1, TRPV2, TRPV3, TRPV5, TRPV6, TRPA1, mCa1,
mCa2),[83-85] a plant ion channel,[86] Ca2+-ATPase,[87] mitochondrial Ca2+
uniporter,[88] tubulin,[89], myosin light-chain phosphatase,[90] and Ca2+ binding
proteins such as calmodulin.[91] Ruthenium red has nonetheless been administered in
vivo to antagonize TRPV4 activated by 4αPDD or other modalities in both the CNS and
the periphery.[64,66,67]
Table 3. Data summary for representative TRPV4 antagonists
Agonist
Compound number
Potency (IC50, M)
Species
Reference
Ruthenium Red RN-1734 RN-9893 Capsazepine Citral GSK205
26 27 28 29 30 40
<1, 0.21 <0.2, 0.33 <1, 0.086 2.3 5.9 3.2 <0.12a <0.06a <0.12a 18.6 13.5 32b
0.6c
mouse rat human human mouse rat human mouse rat human rat mouse pig
[61,69] [14,69] [61,69] [69] [69] [69] [82] [82] [82] [69] [69] [92] [93]
a KB value; b KD value; c Chondrocytes from the indicated species were used for this determination.
RN-1734
RN-1734 27 (Figure 7) was isolated from the same focused library of commercial
aryl sulfonamides as the TRPV4 agonist, RN-1747 15.[69,82] It inhibited the activity of
16
human, rat and mouse TRPV4 with IC50 values in the single-digit micromolar range
(Table 3). Importantly, RN-1734 27 demonstrated an ability to fully antagonize TRPV4
activated by 4PDD, RN-1747 and hypotonicity with similar potencies. Further testing of
RN-1734 in a small TRP channel selectivity panel containing TRPV1, TRPV3 and
TRPM8 revealed it to be selective for TRPV4.
Figure 7. TRPV4 antagonists Ruthenium Red, RN-1734, Capsazepine and Citral
S
Cl
ClO
ON HN
RN-1734 27
N
HO
HO NH
SCl
Capsazepine 29
O
Citral 30
Ruthenium Red 26
RuH2N
H2N NH2H2N
NH2
RuONH2
NH2H2N
H2NRuO
NH2
NH2H2N
H2NNH2 Cl6
RN-9893
Our research group has also identified an orally bioavailable small molecule
antagonist of TRPV4 (named RN-9893 28; Table 3), with KB values <120 nM against the
human, rat and mouse variants of the TRPV4 receptor when stimulated with 4PDD.[82]
Additionally, RN-9893 displays selectivity over TRP family members TRPV1, TRPV3
and TRPM8. In young Wistar rats, RN-9893 is orally bioavailable (%F > 45 %), and has
a moderate half-life (>40 min).[82] However, as of early 2010, the chemical structure for
RN-9893 has not been disclosed.
17
Capsazepine
Capsazepine 29 (Figure 7), a prototypical TRPV1 antagonist, was revealed to also
antagonize TRPV4 in a recent study, albeit at significantly higher concentrations
(approximately 15 M for TRPV4 compared with submicromolar levels for TRPV1).[69]
Awareness of this antagonism, including the sizable differential in potency against these
two ion channels, might potentially be useful for the interpretation of biological data
obtained in complex systems with high concentrations of capsazepine.[94]
Citral
The interaction of citral 30 (Figure 7), a major component of lemongrass, with
multiple TRP channels was recently investigated.[92] Results indicated it was a
reversible TRPV4 antagonist which inhibited channel function by a voltage-independent
mechanism. While citral bound to mTRPV4 most potently with a KD of 32 M, its
activation of TRPV1, TRPV3 and TRPM8 and inhibition of TRPA1 was also
documented.
Antagonists from GlaxoSmithKline (GSK)
GlaxoSmithKline have published a number of patent applications centered around
a diazabicyclo[2.2.1]hept-2-yl chemotype.[95-99] These compounds show structural
similarities to the TRPV4 agonist GSK1016790A 25, and can be regarded as
conformationally-constrained relatives of this compound. Unfortunately, no structure-
activity relationships have been detailed for the GSK TRPV4 antagonists at the present
time (February 2010). Therefore, only a small number of representative compounds from
18
the current patent literature have been detailed below (Figure 8). However, it can be seen
that many substructural features present in the TRPV4 antagonists detailed in Figure 8,
are also present in the TRPV4 agonists shown in Figures 3-6 above. Therefore, it is
possible that the antagonists and agonists detailed by GSK share a similar binding site on
the TRPV4 ion channel.
Figure 8. Representative TRPV4 antagonists from GSK patent applications
19
NN
ONH
SOO Cl
Cl
OHN
ONH
H
H
31
NN
ONH
SOO Cl
Cl
OHN
OS
H
H
32
From WO2009/111680 (>80 examples)
NN
ONH
SOO
ON
H
H
O
S
NH
NN
ONH
SOO
ON
H
H
ONH
Cl
CF3
Cl
CF3
From WO2009/146177 (3 examples)
From WO2009/146182 (>25 examples)33 34
NN
O
OHN
ONH
H
H
35
N
O
Cl
Cl
F
NN
O
OHN
ONH
H
H
36
NS
O O
Cl Cl
From WO2010/011912 (>20 examples)
NN
O
OHN
ONH
H
H37
Cl
CF3
From WO2010/011914 (>200 examples)
NN
O
OHN
ONH
H
H N
N
F
NN
O
OHN
ONH
H
H NH
N
38 39
The structure of GSK205 40, a TRPV4 antagonist based on an amino-thiazole
scaffold, was also published recently.[93] This compound was shown to inhibit TRPV4
in porcine chondrocytes at submicromolar concentrations (Table 3) and was reported “to
block both ligand-gated and hypo-osmotic activation of endogenous and transiently
20
expressed human TRPV4 channels”. Further, GSK205 40 was claimed to be a selective
TRPV4 antagonist, although specific selectivity windows were not provided.
Figure 9. Aminothiazole TRPV4 antagonist, GSK205
N
S
NNH
N
.HBr 40
Patent applications from Kobayashi Pharmaceutical
The vanilloid derivative 41 has been shown to be a TRPV4 antagonist in a recent
patent application (Figure 10).[100] However, the authors of this current review have not
read an English translation of this patent application, so it is difficult to determine any
structure-activity relationships in the small number of compounds evaluated (note:
compound 41 was obtained from the SciFinder summary of this application).
Figure 10. TRPV4 antagonist from Kobayashi Pharmaceutical
NH
MeO
HO
O
C9H1941
21
Selectivity
Selectivity is a key variable in drug discovery, especially when considering a
molecular target in a family containing several closely related members. As could be
expected based upon their sequence homology, and on a similar activation by elevated
temperatures (hot, warm), TRPV1 and TRPV4 are pharmacologically similar to each
other. Examples are numerous and include arachidonic acid derivatives (anandamide,
5,6-EET 12), phorbol esters (RTX, 4PDD 1), as well as compounds capable of
interacting with either channel: RN-1747 15, GSK1016790A 25, ruthenium red 26,
capsazepine 29 and citral 30. Interestingly, a similar, although less obvious relationship
was uncovered between TRPV1 and TRPM8.[101] Considered in this light, the TRPM8
activity displayed by TRPV4 agonist RN-1747 15 and by citral 30 may not be surprising.
Overall, however, large differences in potency are often noticed for compounds binding
to TRPV4 and TRPV1 or TRPM8, indicating that it might be possible to successfully
address selectivity issues.
Conclusion
TRPV4 is a polymodal receptor with a wide expression pattern and a
corresponding variety of physiological roles. Several small molecule agonists, including
4αPDD 1 and GSK1016790A 25, have been discovered and used to study this channel.
In comparison, the development of TRPV4 antagonists has been markedly slower and the
first non-metal based small molecule antagonists have appeared only recently.[69,82,95-
22
99] Their application will likely shed some light on the potential of TRPV4 inhibition for
the treatment of inflammatory and neuropathic pain, bladder and urinary disorders, and
possibly rare genetic disorders linked to TRPV4. The breadth of functions fulfilled by
this channel, however, will ensure that the monitoring of potential on-target toxicity will
be a key part of any drug discovery project targeting TRPV4.
Addendum
TRPV4 antagonist from Hydra Biosciences
After the acceptance of this manuscript, a report emerged describing the profile of a
small-molecule TRPV4 antagonist, HC-067047 42.[102] When dosed at 10 mg/kg i.p.,
HC-067047 improves bladder function in rats and mice with cyclophosphamide-induced
cystitis. HC-067047 is a potent antagonist of the human, rat and mouse orthologs of
TRPV4 (4PDD used as stimulating agonist). It also inhibits TRPM8 and the potassium-
channel hERG at sub-micromolar concentrations (approximate 10-fold window of
selectivity for TRPV4 over TRPM8 and hERG; the hERG activity for HC-067047 may
limit its use to the preclinical setting). Importantly, HC-067047 did not affect core body
temperature, thermal selection behavior, water intake, heart rate, locomotion or motor
coordination in vivo. These last results will be particularly encouraging for those
interested in developing TRPV4 antagonists to address human diseases. In summary, HC-
067047 is a fine addition to the TRPV4 field, and may prove to be a very useful tool with
which to probe the in vivo function of TRPV4 antagonists beyond the bladder indication
described by the present study.
23
Figure 11. TRPV4 antagonist HC-067047
HC-067047 42hTRPV4 IC50 = 48 nMrTRPV4 IC50 = 133 nMmTRPV4 IC50 = 17 nM
active at 10 mg/kg i.p. in model of cystitisN
ONH
NO
CF3
24
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