Carotenuto, Lidia (2023) Identification and pharmacological characterization of novel Kv7 potassium channels modulators for the treatment of hyperexcitability disorders. [Tesi di dottorato]

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Abstract

The Kv7 subfamily of voltage-gated potassium (K+) channels includes 5 members (Kv7.1-Kv7.5) having distinct expression patterns and physiological roles. In neurons, Kv7.2, Kv7.3 and Kv7.5 subunits underlie the so-called M-current (IKM), a sub-threshold K+ current playing a critical role in the control of neuronal excitability. Mutations in the genes encoding for Kv7.2, Kv7.3 and Kv7.5 are responsible for a wide spectrum of early-onset epilepsies. Retigabine is the first antiepileptic drug acting on Kv7 channels. It was approved for clinical use in 2011 as an adjunctive therapy in adults showing drug-resistant partial onset seizures. Retigabine suppresses neuronal hyperexcitability by shifting the Kv7.2/7.3 activation threshold toward more hyperpolarized potentials, thereby increasing their participation to the stabilization of the membrane potential. Unfortunately, retigabine suffers from considerable drawbacks including photo instability: light exposure causes retigabine photodegradation and oxidation, leading to the formation of dimers. Upon long-term use, retigabine dimers accumulate into light-exposed tissues, thus inducing retinal and mucocutaneous blue-gray discoloration in patients. This led to a progressively reduced use of retigabine, until the manufacturing company (GSK) has decided to withdraw the drug from the market in 2017. Since then, no Kv7 activator is clinically available as anticonvulsant. The present work originates from our effort to identify novel and safer Kv7 channels activators to be used as new antiseizures medication. For this purpose, we developed a cellular fluorescence-based assay to rapidly evaluate the effect of Kv7 modulators, suitable for both small-scale and large-scale high throughput screening (HTS). The assay was exploit for two different drug discovery approaches: 1. Structure-based Drug Design: retigabine structure-activity relationship (SAR) and the molecular determinants responsible for its photo-induced dimerization were combined to guide the design of novel analogues. The new-synthesized retigabine derivatives were screened for their Kv7 opening ability using the fluoresce-based assay. Among them, compound 60 was unable to form photo-induced dimers, was more potent and effective than retigabine in activating Kv7.2/7.3 currents in vitro and in vivo as anticonvulsant in an acute epilepsy animal model. 2. Drug Repurposing: the Fraunhofer repurposing library, containing more than 5600 bioactive compounds already tested in preclinical and clinical studies, was screened in search of new Kv7 channel openers using the fluoresce-based assay adapted to a large-scale HTS. From the screening, C4 emerged as the most potent and effective newly identified Kv7 channel opener; further in vitro characterization of C4 revealed an efficacy and potency comparable to retigabine in activating Kv7.2/7.3 currents.

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