EAG1 as a target for cancer diagnosis and therapy in pediatric tumors: from early to late effects.

Cancer is a complex disease in which abnormal cells divide uncontrollably. Cancer cells can spread throughout the body and interfere with the normal function of tissues and organs. It is one of the leading causes of death by disease worldwide and has a significant economic impact. Novel targeted approaches for the treatment of cancer are needed to reduce this high mortality.

Here, we focus on the cancer target Kv10.1 (hEag1). This target is a voltage-gated potassium channel that is overexpressed in a wide range of cancer cells. Inhibition of this channel leads to a reduction of cancer cell proliferation and migration in vitro and in vivo. Although several small molecules and antibodies are known to inhibit this channel, none of them have reached the market so far.

In this PhD project, we screened an array of small molecules, venom fractions and peptide toxins for inhibitory activity on Kv10.1. Although this screening was performed using a low-throughput electrophysiological approach, we were able to identify several novel Kv10.1 inhibitors. This compound screening is described in Chapter 3. We identified the two first peptide toxins known to exert inhibitory activity on Kv10.1, i.e. the scorpion peptide κ-hefutoxin 1 (Chapter 4) and the sea anemone peptide APETx4 (Chapter 5). Finally, we also identified a novel group of small molecules that act on Kv10.1, namely purpurealidin analogs derived from a secondary metabolite of marine sponges (Chapter 6).

κ-Hefutoxin 1 showed the lowest potency (~ 26 µM) of all the identified inhibitors, it is also known as a weak Kv1.x inhibitor. Moreover, a loss of activity and a batch-to-batch variability was observed. Therefore, we believe that this scorpion toxin is not an ideal drug candidate. However, since no other peptides were known before the start of this project, this peptide can be an interesting tool to study Kv10.1 and to design more potent, selective and stable peptides.

APETx4 is one of the most interesting Kv10.1 inhibitors that was identified in this project. This peptide acts as gating modifier and keeps the channel in a closed state, presumably by binding to the voltage paddle. At low micromolar concentrations, APETx4 is able to distinguish between the Kv10.1 channel and the related cardiac channel hERG (Kv11.1). Moreover, the peptide is able to induce cytotoxicity and apoptosis in several cancer cell lines. APETx4 also inhibits other ion channels and is therefore not selective for Kv10.1. In a follow-up project, APETx4 mutants will be synthetized in order to identify more selective peptides that can be lead compounds in the search for novel anticancer drugs.

Finally, we identified a group of sponge secondary metabolite analogs as novel Kv10.1 inhibitors. These purpurealidin compounds act in a completely different manner than APETx4. They also seem to induce a voltage-dependent inhibition, but the steady-state activation curve is now shifted to more negative potentials. Whereas upon APETx4 application, the steady-state activation curves shift to more positive potentials. The sponge compounds seem to induce an apparent inactivation and they accelerate the kinetics of current activation. This effect is reminiscent of that of mibefradil, which modifies the gating characteristics of Kv10.1. Unfortunately, the most potent analog also inhibits the cardiac hERG channels and induces a cytotoxic effect in cancerous and healthy cell lines.

To conclude, we identified several new Kv10.1 inhibitors. These inhibitors can be used as tools to study the cancer-related potassium channel Kv10.1 and/or as templates for the design and synthesis of novel anticancer drugs.