TECHNOLOGY PLATFORMS FOR THE STUDY OF ION CHANNEL BIOLOGY AVAILABLE AT REACTION BIOLOGY CORP

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1 TECHNOLOGY PLATFORMS FOR THE STUDY OF ION CHANNEL BIOLOGY AVAILABLE AT REACTION BIOLOGY CORP Introduction Ion channels are integral membrane proteins that mediate the regulated passage of charged particles (ions) across otherwise impenetrable membranes. Most ion channels are multi-subunit protein complexes that assemble in such a way as to enable the formation of a water-filled pore through lipid bilayer membranes through which ions flow along their electrochemical gradients. Electrochemical gradients are defined by the relative concentration gradients of individual ions across the cell membrane and the transmembrane electrical potential. Ion channels control basic cellular functions including establishment of the resting membrane potential, regulation of cell volume control, control of ion and water flow through a variety of secretory and epithelial cells as well as the control of electrical signaling including action potential initiation, shaping and termination - in electrically-excitable cells (neurons, cardiomyocytes ). There are over 400 identified ion channel subunits that assemble in an essentially combinatorial manner allowing for tremendous diversity in structure and function across this gene class. Ion channels are critical molecular targets for both drug discovery and safety pharmacology. Prescribed medicines that target ion channels directly or indirectly are estimated to account for > $20 billion annually in expenditures for medicines worldwide and represent ca. 15% of FDA-approved drugs (second only to GPCR-directed drugs as a class). Reaction Biology Corporation has recently added ion channel biology capabilities to our catalog of service offerings. Test platforms implemented and validated include Sophion QPatch work stations, traditional manual patch clamp, Two Electrode Voltage Clamp (TEVC) using Xenopus oocytes and fluorescence-based ion channel methodologies (Hamamatsu FDSS6000). In addition to having the instrumentation required to address all ion channel testing needs, Reaction Biology scientists have extensive experience in all aspects of these testing platforms. Recent screening campaigns have

2 included multiple screens monitoring calcium flux using the FDSS6000 system for four separate NMDA ion channels. Follow-up activities included evaluating compound activities using automated patch clamp in the QPatch as well as compound profiling against multiple ionotropic glutamate channels using the TEVC platform. Finally, compound activities can be assessed in primary neuronal cultures using the manual patch clamp system. In addition, the pharmacologists at Reaction Biology have established validated testing processes for ion channels critical for cardiac safety studies including herg, Nav1.5 and Cav1.2 assays using the QPatch as well as manual patch clamp technologies. Patch Clamp Electrophysiology: Manual and Automated It is generally acknowledged that manual patch clamp is the gold standard technology for studying the biophysical properties of ion channels. Manual patch clamp technologies deliver highly precise, information-rich recordings of ion channel activities with resolution from the whole cell level to the single channel recordings. However, practical limitations of the technologies have largely precluded widespread adoption in most laboratories. These limitations include the absolute requirement for highly trained/highly skilled operators and the low testing throughput characteristic of the process. To help mitigate these limitations, several companies have developed more automated patch clamp technologies and instrumentation platforms some of which are capable of delivering high fidelity recordings that rival the high quality of manual patch clamp recordings while delivering significantly higher levels of testing throughput. These technological advances have largely overcome the limitations noted for manual patch clamp recordings including the need for highly specialized operators. Reaction Biology Corp now offers both manual patch clamp and automated patch clamp testing providing a broad base of ion channel testing capabilities for our clients. Below are representative experimental results taken from assays characterizing the biophysical properties of the herg cardiac voltage-gated potassium channel (human ether-a-go-go-related gene; Kv11.1) in both manual patch clamp and automated patch clamp platforms. The automated patch clamp data are taken from our Sophion QPatch HTX instrument (Sophion Bioscience A/S, Ballerup, Denmark). The herg channel expressing cells used in these experiments were obtained from B SYS GmbH (Witterswil, Switzerland) CHO herg-duo cell line. The herg channel is responsible for the delayed rectifier rapid (IKr) current that is a key component of the cardiac ventricular myocyte depolarization/repolarization cycle. The herg channel has emerged as a critical target for safety pharmacology. In fact, all new medicines are required to undergo profiling against the herg channel in electrophysiology assays in order to secure FDA approval. Figure 1A (manual patch clamp) and 1B (automated patch clamp; QPatch) demonstrate representative currents elicited by step depolarization to potentials Figure 1. Current vs. time (I/t) tracings for activation of herg channels. Original whole cell current tracings of voltage-gated herg currents in manual patch clamp (A) and QPatch (B) platforms. See text (above) for descriptions of the voltage protocols used. A: B:

3 between -60 and +40 mv (manual patch) or -80 and +50 mv (QPatch) from a holding potential of -80 mv. In both cases, tail currents were recorded after a step repolarization back to -50 mv. The elicited voltagedependent current traces are shown in Figures 2A (manual patch clamp) and 2B (QPatch). Outward currents were activated at voltages positive to -50 mv with peak steady state currents observed at +10 mv (manual patch clamp) and -10 mv (QPatch). In both cases, internal rectification is apparent at more positive voltages. The normalized peak tail current data were fitted to a Boltzmann function shown in Figures 2C (manual patch clamp) and 2D (QPatch). The half maximal activation voltage (V1/2) and slope factors (k) were -9.7 mv (k = 11.4) and mv (k = 8.7) for manual patch clamp and QPatch, respectively. Figure 2. Current-voltage (I/V) and activation plots. Current activation plots for the steady state herg currents recorded via manual patch clamp (A) and QPatch (B), respectively. Normalized peak tail current/voltage plots recorded via manual patch clamp (C) and QPatch (D), respectively. The effect of terfenadine (a known blocker if I Kr ) on the amplitude of herg outward currents in the QPatch is shown in Figure 3. Terfenadine caused a concentration-dependent decrease in the peak herg tail currents with an IC50 of 42 nm. The potencies of additional known herg blockers are presented in Table 1. Figure 3. Blockade of herg currents be terfenadine recorded in the QPatch. Table 1. Reference compound inhibition of herg peak tail currents A: Concentration-dependent inhibition of herg currents. Compound IC 50 (nm) terfenadine 42 amitriptyline 2800 dofetilide 6 quinidine 76 cisapride 39 mexiletine >30000 verapamil 420 B: Hill plot of the inhibition of normalized tail current data Additional details on our manual patch clamp and automated QPatch platforms are available upon request.

4 Additional details on our manual patch clamp and automated QPatch platforms are available upon request. Two-Electrode Voltage Clamp (TEVC) in Xenopus laevis Oocytes Xenopus oocytes are routinely used as a robust expression system for functional ion channel current measurements. The large size of the oocytes allows for the direct injection of cdnas or crnas leading to reliable functional expression of target proteins. In TEVC, the plasma membrane of the oocyte is impaled by two microelectrodes one of which constantly monitors membrane potential while the second electrode injects current to maintain the membrane potential at a given set point (e.g., -40 mv) as dictated by a feedback amplifier circuit. The following example of a TEVC recording session (Fig.4) demonstrates the effects of a Positive Allosteric Modulator (PAM) of NMDA currents (NR1/NR2D subtype) on an oocyte co-injected with NR1 and NR2D channel subunits ca. 72 hours prior to recording. In this experiment, the oocyte is exposed to increasing concentrations of the PAM in the presence of saturating concentrations of the coagonists glutamate and glycine. The Hill plot of the data from the cumulative concentrationresponse curve is shown. Fluorescencebased Ion Channel Assays Fluorescence-based assays are widely used, especially for applications needing higher levels of throughput. Unlike the true electrophysiology Figure 4. TEVC tracing from NR1/NR2D potentiator assay. (A) Original data recording of potentiation of NR1/NR2D current by test compound in the presence of saturating co-agonists glutamate and glycine. (B) Hill plot of potentiation data. A: B: Figure 5. Fluorescent traces of calcium influx in RINm5f cells loaded with Fluo8 AM and exposed to increasing concentrations of AITC measured in the FDSS6000. RINm5f cells were loaded with 5 µm Fluo8 AM with probenecid (2.0 mm) for 60 minutes at room temperature. Medium with unabsorbed dye was removed and the cells subsequently exposed to test agents in the FDSS6000. Results shown are the fluorescent traces from individual wells exposed to the agonist AITC at several concentrations as indicated in the fluorescence trace.

5 assays described above, fluorescence-based assays do not directly measure ion currents. Fluorescencebased assays utilize intracellular probes either chemical or genetically-encoded that monitor specific ion fluxes or measure changes in membrane potential as a result of depolarization/hyperpolarization. In the example shown below, RIN-m5f cells (rat pancreatic beta cell insulinoma) were loaded with the calcium indicator dye, Fluo8 AM, and incubated with chemical agents known to interact with TRPA1 channels. Raw fluorescent traces (FDSS6000) of activator data are shown in Fig. 5. Activators of TRPA1 channels used in these experiments included allyl isothiocynate (AITC, Fig. 6A), trans-cinnamaldehyde, icilin, and polygodial (not shown). Antagonists included A and HC (Fig. 6B). Fluorescent assays that monitor calcium flux are the most commonly used fluorescence-based assays for ion channel activity. These assays are commonly conducted to monitor activity of voltage-gated calcium channels as well as activity of ligand-gated ion channels with non-selective cation conductances. Figure 6. Hill plots of agonist and antagonist concentration-response curves. (A) Agonist response to AITC. (B) Antagonism of AITC-induced calcium mobilization by A and HC A: B: Reaction Biology Corp will be expanding our offerings in cell signaling to include new ion channel screening capabilities including the abilities to monitor potassium and chloride channel flux using appropriate chemical and genetically-encoded sensors for the surrogate channel markers thallium and iodide, respectively. In addition, Reaction Biology will be adding to our catalog of GPCR-relevant assay formats to include the abilities to monitor second messenger generation including camp and beta-arrestin dynamics in addition to established technologies such as calcium mobilization. Contact us today and learn how Reaction Biology scientists can accelerate, enhance the value of and bring innovation to your discovery activities. sales@reactionbiology.com