DESIGN AND PRECLINICAL DEVELOMENT OF CP86, A NEW POTENT IN VIVO ANTIEPILEPTIC AGENT

Kv7.2/7.3 potassium channels represent an attractive pharmacological targets for the treatment of different neurological disorders, in particular epilepsy. During 2011 the Kv7.2/7.3 agonist retigabine has been approved as add-on treatment for drug-resistant partial onset seizures with or without secondary generalization. However, the clinical use of retigabine has been declining over the years, leading to its market withdrawal in 2017,1 due to several drawbacks and side effects. The resulting limitation in therapeutic options led to extensive efforts, both from academia and industry, in the search for new Kv7.2/7.3 agonists. These efforts were largely frustrated by a substantial lack of knowledge concerning retigabine binding site.1 This is why, starting from our previously published results,1 we have performed in-silico studies to shed light on the chemical space at the retigabine binding site. A structure-based approach was used to verify the in-silico hypotheses synthesizing a focused library of 25 compounds, which were tested by an HTS fluorometric assay. Results obtained further expanded the structure-activity relationship clues for the rational design of Kv7.2/7.3 channels agonists, confirming the pivotal importance of a wide, lipophilic pocket in correspondence of the pore region for the modulation of Kv7.2/7.3 agonists potency and efficacy. Nevertheless, as assessed by photostability testing, synthesized compounds were unable to overcome one of the main retigabine drawback: light-induced instability. Thus, taking advantage form the previously obtained SAR clues, a photostability-driven design was performed, generating a second set of 16 molecules. Among these, CP86 showed improved potency, efficacy and photostability when compared to retigabine. Patch-clamp experiments confirmed these preliminary data. CP86 is able to produce a marked leftward shift of the dose response curve and increased maximal currents in comparison to Kv7.2/7.3 prototypical agonists. Site-specific mutagenesis experiments validated the predicted binding mode, involving an interaction network with W236, V225, F240, S303, F304, F305 and L312, in the Kv7.2/7.3 subtype heterotetrameric assembly. CP86 was, then, subjected to an extensive in vitro and in vivo preclinical characterization resulting as a metabolically stable compound, with considerably improved half-life and CNS distribution if compared to retigabine. When challenged in vivo, by a pentylenetetrazol (PTZ) kindling model of epilepsy, CP86 showed a remarkable reduction of the incidence and severity of tonic PTZ seizures, at one-twelfth of the retigabine dose. Moreover, seizures modulation was combined with an outstanding protective effect. CP86 administration, indeed, largely prevented pentylenetetrazol-induced death, widely described2 and experimentally observed for retigabine-treated animals