Ion Channel Retreat Channel Ion 2014 June Vancouver, Background
• A division of SB Drug Discovery
• Located in Glasgow, Scotland
• Established in 1994
• 25 researchers Overview
• SB Drug Discovery specializes in protein expression, cell line development and assay services
• Developed > 100 custom cell lines expressing ion channels, GPCRs, antibodies etc
• Developed Nav1.1 – Nav1.8 sodium channel panel
• Highly experienced electrophysiology team
• SB Ion Channels Division set up in 2013
Off the shelf ion channel cell lines
Custom ion channel cell line development
Ion channel screening services SB Ion Channels
• Parallel processing systems put in place to enable efficient development of multiple ion channel cell lines simultaneously
• Last 12 months : 40 ion channel cell lines developed
• Currently offering over 50 ion channel targets
• Further 6 targets in development Voltage gated channels
Sodium Potassium Other Channels Channels Channels Nav1.1 Kv2.1 Kir2.1 Ano1
Nav1.2 Kv2.1 / 9.3 Kir2.2 Ano2
Nav1.3 Kv2.2 Kir2.4
Nav1.4 Kv3.1 SLACK
Nav1.5 Kv3.2 TREK-1
Nav1.6 Kv7.2 TRESK
Nav1.7 Kv7.2/7.3
Nav1.8 Nav1.7 monkey Nav1.7 dog
Nav1.7 rat Ligand gated channels
Purinergic TRP Channels Other Channels Channels
P2X1 TRPA1 TRPV1 TRPM2 TRPC1 NMDA TRPA1 P2X2 TRPV2 TRPM3 TRPC3 monkey TRPA1 P2X3 TRPV3 TRPM4 TRPC4 sheep P2X4 TRPV4 TRPM5 TRPC5
P2X5 TRPV5 TRPM8 TRPC6
P2X6 TRPV6 TRPC7
P2X7 TRPV1 rat
P2X7 mouse TRPV1 dog (transient) Pain related channels
Pain related channels available at SB Nav1.1 TRPV3 TRPC4 Kv7.2/7.3 Nav1.2 TRPV4 TRPC5 TREK-1 Nav1.3 TRPM2 TRPC6 TRESK Nav1.7 TRPM3 P2X1 SLACK Nav1.8 TRPM4 P2X2 Ano1 TRPA1 TRPM8 P2X3 NMDA TRPV1 TRPC1 P2X4 TRPV2 TRPC3 P2X7
Animal variants Dog Nav1.7 Monkey TRPA1 Rat TRPV1 Dog TRPV1 Rat Nav1.7 Sheep TRPA1 (transient) Monkey Nav1.7 Validation
QPatch - voltage gated
Nav1.1 – Nav1.8 TREK-1 TRESK
Kv3.1 Ano 1 Ano 2 Validation
QPatch – ligand gated
TRPM4 TRPM5 TRPA1 TRPA1 (monkey)
Awaiting data analysis
TRPV1 TRPV3 TRPV4 TRP channels
• 22 TRP channels
TRPA1 TRPA1 (monkey) TRPA1 (sheep) TRPC1 TRPC3 TRPC4 TRPC5 TRPC6 TRPC7 TRPM2 TRPM3 TRPM4 TRPM5 TRPM8 TRPV1 TRPV2 TRPV3 TRPV4 TRPV5 TRPV6 TRPV1 (rat) TRPV1 (dog)
• Customized selectivity panels
• Flexstation assay or manual patch clamp assessment
• Ongoing development of single use, assay ready cells for custom selectivity kits Moving forward
Development of 30 new ion channel cell lines
Targets selected based on client feedback
Optimized cell lines for QPatch & IonWorks
Custom Cell Line Development
• Conventional cell line development process
• Risk-free development
• Gene synth, subcloning, mutagenesis Molecular • Sequence analysis Milestone 1 Biology
Deliverable – sequence validated expression plasmid • Transfection, clonal selection Cell • Single cell colony growth Biology
• Colony screening • Detailed electrophysiology assessment Validation • Stability, mycoplasma, stock generation
Milestone 2
Deliverable – Functionally validated stable cell line (defined criteria) Improving cell lines
Sub-cloning
300000 I (pA) 250000 Na
200000 mV -100 -80 -60 -40 -20 20 40 60 80 100 150000
RFU RFU -1000 100000 -2000 50000 Parental Control Parental TTX 100 nM -3000 0 Subclone Control Subclone TTX 100 nM -4000 HEK UT HEK CLONE17 CLONE68 CLONE91 CLONE92 CLONE93 CLONE94 CLONE95 CLONE96 CLONE97 CLONE98 CLONE99
CLONE100 CLONE101 CLONE102 CLONE103 CLONE104 -5000 PARENTAL CLONE Parallel Processing
1 2 3 4 5 6
Fold increase in time Fold increase in cost versus single cell line versus single cell line
0 Molecular Biology 4-fold
Transfection & 0 2-fold selection
0 Colony screening 2-fold
3-fold Functional 6-fold Characterization
<1.5-fold <3-fold Parallel Processing
Development of multiple cell lines in parallel
• Selectivity panels
• Animal variants
• Disease-related mutants
• Mixed selections
Benefits
• Minimal increase in timelines
• Significant cost savings
Assay services
• Flexstation, conventional electrophysiology, IonWorks, QPatch
High throughput screening Selectivity profiling Biophysical characterization Lead optimization
• DRG studies
• In vivo models of pain DRG studies
Freshly isolated rat DRG
Presence of sodium, calcium, potassium & TRP channels
Potassium currents
6000 1000
4000 0
2000
-1000 0
-2000 Calcium currents Sodium currents
2000
1500
1000 Delayed rectifier currents
500
0 DRG studies
TRPs channels in DRGs cells
100 CurrentCurrentPeakPeak (pA) (pA)
0 Menthol 500 µM AITC 100 µM (TRPM8) Capsaicin 100 µM (TRPV1) (TRPA1)
0 10 20 30 40 50 60 70 80 Voltage Ramps (n) Proof of concept in vivo models of pain
Inflammatory pain (CFA)
• Complete Freund’s adjuvant injected into hind paw • Acute & sub-chronic inflammation at 24hr / 7 days respectively
• Inflammatory cascade causes swelling & pain lasting 1-14 days
• Thermal hyperalgesia (Hargreaves plantar test) & mechanical hypersensitivity (weight bearing) assessed
55.0
** ** 16 * 50.0 ** * ** ** * ** 45.0 ** vehicle 12 * 3 mg/kg 10 mg/kg 40.0 30 mg/kg
8 % weight bearing 35.0 Paw withdrawal latency (s)
4 30.0 Baseline 0 1 3 5 Baseline 0 1 3 5 Time (h) Time (h)
Carrageenan model also available Proof of concept in vivo models of pain
Neuropathic pain (Chung model)
• Tight ligation of the L5 spinal nerve • Mechanical allodynia measured by paw withdrawal threshold (von Frey filaments)
• Thermal hyperalgesia measured by Hargreaves plantar test
Vehicle 4 mg/kg 15.0 *** 10 mg/kg 40 mg/kg
*** *** ** 10.0 ***
**
5.0 Paw Paw thresholdwithdrawal (g)
0.0 Baseline 0 0.5 1 2 3 Time (h)
Bennett (CCI) model also available Proof of concept in vivo models of pain
Post-operative pain (Brennan model)
• Incision made in plantar surface of hind paw, plantaris muscle elevated & wound closed • Causes spontaneous pain, thermal & mechanical thresholds reduced for 4 days • Thermal hyperalgesia (Hargreaves plantar test) & mechanical allodynia paw withdrawal (von Frey filaments) assessed
** 25 ** 15 ** ** **
20 ** ** ** 10 vehicle 15 * 0.3 mg/kg ** 1 mg/kg 3 mg/kg 10 6 mg/kg 5
5 Paw withdrawal latency (s) Paw withdrawal threshold (g)
0 0 Baseline 0 0.5 1 2 Baseline 0 0.5 1 2 Time (h) Time (h) Proof of concept in vivo models of pain
Acute nociception tests
• Measure response thresholds to high intensity stimuli • Hotplate & tailflick tests in response to thermal stimulus in vivo expertise
• In vivo specialists are SB Drug Discovery employees
• All work carried out at SB Drug Discovery’s site
• Scientists possess the relevant animal licences
• Each specialist has >10 years in vivo experience
• SB standard protocols or client specified methods
• All models are validated and ready to run Summary
Extensive panel of off-the-shelf ion channel cell lines
Largest commercial source of TRP channels
Risk -free ion channel cell line development
High throughput screening, profiling & biophysical characterization
DRG studies & in vivo models of pain