Screening for State-dependent Blockers of
Voltage Gated Calcium Channels
P08
Margaret S. Lee , Glenn Short , Terrance P. Snutch
1
1Zalicus
Voltage Gated Calcium Channel States
Pathological pain is widely considered to be a hyper-excited signalling state. Numerous lines of evidence, including
gene c and pharmacologic interven on, have implicated both N-type and T-type voltage gated calcium channels in
the biology and pathophysiology of pain signalling. During normal signalling, voltage gated calcium channels cycle
through various ga ng states in response to membrane poten al changes which in turn alter calcium flux through the
channel. With prolonged excita on, such as during chronic pain signalling, the propor on of channels in inac vated
state increases due to slow recovery kine cs that prevent the ion channel from rapidly rese ng to the closed state.
This natural “braking” feature of pain signalling offers an opportunity for pharmacological selec vity through
specifically targe ng the inac vated state. By specifically targe ng the inac vated state it may be possible to
broaden therapeu c window, minimize adverse effects and increase efficacy. To this end, we have established cell
based assays for state dependent modula on of na ve and recombinant calcium channel func on. Compara ve
screening of channels in closed vs. inac vated states, has resulted in the discovery of novel, first in class calcium
channel blockers that demonstrate enhanced potency for the inac vated state. Z160 is a selec ve N-type calcium
channel blocker, which mediated a hyperpolarizing shi in steady-state inac va on, consistent with a mechanism
involving inac vated state blockade. Z160 consequently demonstrated use-dependent block of N-type channels, with
significantly greater potency at s mula on frequencies similar to the ac on poten al firing rate typical of neurons
processing pain signals. A er oral administra on in the Chung and Chronic Constric on Injury rodent models of
neuropathic pain, Z160 demonstrated dose-dependent reduc on of thermal hyperalgesia and tac le allodynia at
levels comparable to morphine, ω-conotoxin MVIIA and gabapen n with no motor effects as measured by rotarod.
Z944, iden fied using a high-throughput fluorescence based assay for T-type calcium channel inac va on and a
ra onal structure-based design approach, was observed to inhibit currents from the CaV3.1, CaV3.2 and CaV3.3 Ttype isoforms with sub-micromolar potencies while maintaining selec vity for the inac vated state over the closed
state. Following oral administra on in the Complete Freund’s Adjuvant (CFA) model of inflammatory pain, Z944
caused a significant, dose-responsive reversal of mechanical hyperalgesia which was 2-fold greater than naproxen,
with no seda ve effects. Likewise in formalin models of acute and inflammatory pain, pre-treatment of Z944 in rats
resulted in a significant decrease in the rate of flinch responses during the inflammatory pain phase and in mice
during both the acute and inflammatory pain phases. These data demonstrate the u lity of inac va on state
screening to iden fy novel, potent, selec ve and state-dependent calcium channel blockers with efficacy in animal
models of inflammatory and neuropathic pain. Based upon the pharmacological profile and nonclinical efficacy of
Z160 and Z944, further inves ga on of these small molecules in human subjects is warranted.
rCaV2.2/Kir2.3 State-Dependent FLIPR Assay
MKH buffer
Closed
Read
Baseline
Closed-state
inhibition
Read
Signal
•
•
•
•
•
Open
Read
Baseline
83.0 mM KCl
Read
Signal
Load cells with Fluo-4 loading dye
Wash cells plate with KCl buffer
Add compound and incubate
S mulate with high external K+.
Read fluorescent signal
Fluo-4-AM
(45 min)
(hyperpolarised
format)
2 mM K+ Fluo data acquisition buffer
Test
compounds
(20 min)
Read
Baseline
Closed
7.6 mM K+ Fluo data acquisition buffer
Test
compounds
(20 min)
Fluo-4-AM
(45 min)
Read
Signal
Open
Closed
Fluo loading buffer
Inactivatedstate
inhibition
6.0 mM KCl
Read
Baseline
10.4 mM KCl
Read
Signal
•
•
•
•
•
Load cells with Fluo-4 loading dye
Wash cells with KCl buffer
Add compound and incubate
S mulate with high external K+
Read fluorescence on FLIPR
(depolarised
format)
Closed
Open
100
Example Hit
Closed
Inactivated
Percent Inhibition
80
10 - 6
10 - 7
60
IC50 = 3250 nM
IC50 = 107 nM
40
20
0
10 - 5
10 - 9
10 - 8
10 - 7
Z160 (M)
10 - 6
10 - 5
Methods: The N-type calcium channel was stably expressed in HEK cells and seeded in 384-well plates 48 hours prior to assay. The assay
u lizes an inwardly rec fying potassium channel, Kir2.3, with extracellular potassium concentra ons to control cellular membrane poten al.
Channels are converted from either the res ng state or the depolarized state (~30% of inac vated) and subsequently ac vated by
applica on of high extracellular potassium. Quadruplicate data points are averaged and logis c fit was used to calculate IC50 values.
10-6
10-7
-8
10 -8
10 10-7 10-6 10-5
FLIPR IC50 (M)
Z944
10-5
10-6
10-7
10-8 -8
10 10-7 10-6 10-5
FLIPR IC50 (M)
100 Ca 3.2
V
80
60
*
O
*IC50= 160 nM
IC50= 540 nM
O
40
1 nA
10 ms
-30 mV
20
-75 mV
-110 mV
0
10 ms
10-8
10-7 10-6
Z944 (M)
10-5
Methods: T-type calcium channel subtypes were stably expressed in HEK cells and seeded in 384-well plates 48 hours prior to assay.
Calcium dependent fluorescence measurements were performed using Fluo-4 acetoxymethyl ester (Invitrogen F14202) and Pluronic-F127
(Invitrogen P6867) and the FLIPRTETRA (Molecular Devices) at an illumina on wavelength of 470-495 nm and emission wavelength of 515-575
nm. Detailed methods can be found in Belarde , et al. 2009, Assay and Drug Development Technologies, Vol. 7, p. 266.
Z944 Profile
0.1
1
10
100
20
10
0
***
**
Z160 Dose (mg/kg)
m
g/
kg
0
30
30
20
p<0.0001;
p<0.001 vs. vehicle
One w ay ANOVA, Tukey's post test
Methods:
Male Sprague-Dawley rats 200 to 300 grams at me of tes ng were used. Spinal nerve liga on (SNL) injury was induced using the procedure
of Kim and Chung, (Kim and Chung 1992). Sham control rats underwent the same opera on and handling as the experimental animals, but
without SNL. CCI was induced using the procedure of Benne and Xie (1988). Peripheral inflamma on was induced in the hind paw or the
rat by injec ng 0.1 mL of a 2% 8-carrageenan suspension into the subplantar surface of the hind paw of lightly ether-anesthe zed rats. The
assessment of tac le allodynia consisted of measuring the withdrawal threshold of the paw ipsilateral to the site of nerve injury in response
to probing with a series of calibrated von Frey filaments. Withdrawal threshold was determined by sequen ally increasing and decreasing
the s mulus strength (“up and down” method), analyzed using a Dixon non-parametric test (Chaplan et al. 1994), and expressed as the
mean withdrawal threshold. The method of Hargreaves and colleagues (Hargreaves et al. 1988) was employed to assess paw-withdrawal
latency to a thermal nocicep ve s mulus. A radiant heat source was ac vated with a mer and focused onto the plantar surface of the
affected paw of nerve-injured or carrageenan-injected rats. Data were converted to % an hyperalgesia or % an nocicep on by the
formula: (100 x (test latency - baseline latency)/(cut-off - baseline latency) where cut-off was 21 seconds for determining an hyperalgesia
and 40 seconds for determining an -nocicep on.
Vehicle
Morphine (6 mg/kg sc)
Z944 (30 mg/kg po)
**
g/
kg
Chung - Thermal
Chung - Von Frey
Chung (sham) - Von Frey
CCI - Cold
Carrageenan - Thermal
Rotarod
40
***
40
m
60
***
50
30
80
Formalin (rat)
na
pr
ox
in
100
CFA (rat)
60
g/
kg
Percent Activity on Model Behavior
Z160 Effects in Pain Models
m
Chung (rat)
3
Z160 Profile
Inac vated
10-5
Z9
44
10 - 7
10 - 6
IC50 Closed (M)
Closed
Z9
44
10 - 8
10 - 8
Open
30 %
inactivated
Patch-clamp
Estimated Kd (M)
FLIPR Screen
10 - 5
Paw Withdrawl Threshold (g)
30 %
inactivated
ve
hi
cl
e
Closed
Inhibition (%)
((depolarised
depolarised
fformat)
ormat)
IC50 Inactivated (M)
This natural “braking” offers opportunity
for pharmacological selec vity.
CaV3.2 T-type State-Dependent FLIPR Assay
Fluo loading buffer
12.5 mM K+ EB buffer
Test
compounds
(20 min)
Fluo-4-AM
(45 min)
78.8 mM KCl
Closed
MKH buffer
IInactivatednactivatedstate
s
tate
iinhibition
nhibition
With prolonged excita on the propor on
of channels in inac vated state increases.
2 mM K+ EB buffer
Test
compounds
(20 min)
Fluo-4-AM
(45 min)
Zalicus Compounds
Patch-clamp
Estimated Kd (M)
((hyperpolarised
hyperpolarised
fformat)
ormat)
2
Inc., Cambridge, MA, USA, 2Michael Smith Laboratories, University of Bri sh Columbia, Vancouver, BC, Canada
Abstract
Closed-state
C
losed-state
iinhibition
nhibition
1
Methods:
The Complete Freund’s Adjuvant model of inflammatory
pain was performed as described (Randall 1957) Paw
withdrawal thresholds to a mechanical s mulus,
measured using the Randall-Seli o paw pressure
apparatus, 24 hours following intraplantar injec on of the
adjuvant into a hind paw, were used to calculate the
percent reversal of hyperalgesia. The formalin model was
performed as described (McNamara 2007; Tjolsen 1992).
The number of flinches in Sprague Dawley rats and the
licking response in CF-1 mice was used to measure the
pain score following intraplantar injec on of formalin.
Formalin (mouse)
Vehicle
Z944 (60 mg/kg ip)