Developing novel tools for the detection and modulation of ion channels

Pain is a vital sensory function however when its associated pathways become damaged chronic pain occurs. Major contributors to this pathological process are ion channels, providing a rationale for targeting them therapeutically. The development of detection and therapeutic reagents against ion channels has proved challenging. With the lack of selective chemical probes, most researchers are now studying biological reagents for their ability to target ion channels. These include antibodies and toxins. However, antibodies and toxins have disadvantages of their own, for example they can be difficult to produce. As a result, Affimers may present as an alternative for studying ion channel function. Ion channels are difficult to produce in a format appropriate for isolating binding reagents against. As a result, a number of methods have been developed in an attempt to produce recombinant ion channel proteins suitable for such applications including expression of ion channel extracellular domains, peptide mimotopes and cell-based screening methods. This study investigates a number of methods for presenting ion channels for isolation of Affimer reagents by phage display. Affimer reagents that were able to detect the ion channels TRPV1 and P2X3 in their native cellular context were isolated and characterised by biophysical and cellular assays demonstrating the ability to isolate Affimer reagents capable of binding to and modulating ion channel function. In addition, this study provides a proof-of-concept result demonstrating the use of Affimer reagents in aiding the discovery of small molecule mimetics. The identified molecules demonstrated the ability to modulate TRPV1 when tested in vitro. In conclusion, this study describes a number of methods that have been used to isolate Affimer reagents that are able to detect and modulate ion channels involved in chronic pain.