Ion channels have proven value as drug targets, but the function of many potential targets in this class is unknown. Traditional approaches to elucidating their function, and blocking or enhancing the activity of the channel proteins, have mainly used natural compounds or genetic means, which are laborious, slow and expensive. Reporting in Nature Biotechnology, Xu et al. present a method to rapidly develop ion-channel inhibitors based on a new systematic antibody design strategy.
An obvious key to developing an effective inhibitory antibody is to direct it to the right spot on its target protein. In their report, the authors tested the idea that pattern recognition in the commonly used Kyte–Doolittle (KD) hydrophobicity analysis of the amino-acid sequence provides a simple, readily accessible and effective basis for identifying target sequences in established as well as uncharacterized ion channels.
On a KD plot, ion-pore-forming channel subunits, such as those comprising the voltage-gated potassium channel family or the transient receptor potential (TRP) calcium channel family, show six predicted membrane-spanning segments (S1–S6). The retrospective analysis of the binding sites of existing inhibitory antibodies led the authors to identify a common localization to the E3 domain, a hydrophilic region situated between S5 and S6 — close to the predicted ion pore. The hydrophobicity pattern allows the identification of E3, but the actual peptide sequence is diverse between different proteins, suggesting that highly specific inhibitory antibodies can be developed.
To test this hypothesis the authors generated polyclonal antibodies that were targeted against the E3 region of four different ion channels, including a TRP- and even a voltage-gated Na+ channel, which shows greater complexity compared with voltage-gated potassium channels or TRP calcium channels. For all channels the authors managed to produce highly specific antibodies on first attempt that were capable of blocking 50–60% of the respective ion current.
These results suggest that conventional KD plots can successfully predict hot spots for antibody targeting to create ion-channel inhibitors, to provide new specific tools for linking genes to function and possibly for therapeutic uses. Other patterns on KD plots are evident in more distantly related proteins, such as ligand-gated ion channels and transporters, and should provide the foundations for related antibody strategies. These findings could pave the way for the wider adoption of antibody strategies for therapeutic drug development, with the potential to add to the successes of current antibody-based immuno-modulatory and anticancer therapies.
ORIGINAL RESEARCH PAPER
Xu, S-Z. et al. Generation of functional ion-channel tools by E3 targeting. Nature Biotechnol. 23, 1289–1293 (2005)