A group from The Mount Sinai Hospital / Mount Sinai School of Medicine have identified and characterized a novel a GIRK1-selective activator, GiGA1 capable of selectively opening a subset of G-protein-gated inwardly rectifying K+ (GIRK) channels to produce an antiseizure effect in animal models.
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This study presents an integrated approach to GIRK1-specific activators, employing both computational modeling and subsequent biochemical and physiological studies. The resultant seizure mitigation produced in an acute epilepsy mouse model demonstrates a potential role for this compound in the treatment of brain disorders.
The role of heterotetrameric potassium channels in the onset of a seizure
Four mammalian GIRK channel subunits comprise heterotetrameric channels. The subunit combinations found in the brain are localized and expressed differently. Previous studies employing high throughput screening have identified putative GIRK modulators; however, the lack of subtype-specific compounds has prevented the understanding of the physiological role of region-specific GIRK channels in the brain.
GIRK channels have been implicated in several diseases including Down syndrome, Parkinson's disease, and hyperkinetic movement disorders. Through several mutational studies, GRK channels have proven to play an essential role in the maintenance of the resting membrane potential.
Identifying a potassium channel activator GiGA1
While predominantly activated by G-protein-coupled receptors, the GIRK channels may be GPCRs independent, capable of direct activation by alcohols. Mutagenesis and alcohol tagging studies have underscored the significance of an allosteric binding pocket, characterized by its role in channel activation.
Based on these previous discoveries, the group approached the rational design of small molecules targeted to the alcohol binding pocket, capable of modulating GIRK channel activity.
GiGA1 was identified following a screen of two small chemical libraries totaling ~771,000 compounds using docking programs to conduct virtual screening of alcohol binding site. The 1,000 top-scoring compounds were further subjected to visual inspection to mitigate against the inclusion of false positives.
Zhao et al. constructed a structural model of the alcohol binding pocket present in GIRK2. This was subject to virtual screening; to identify small molecules capable of binding the allosteric site, Zhao et al. virtually screened a fragment-like subset of a small-molecule library database.
A total of 22 compounds were identified for screening against the allosteric GIRK2 pocket. Of those, a so-called “compound 2” produced the most significant activation and was selected for further experiments.
GiGA1 application reduces seizures
The methyl positions of Compound 2 were varied and this effect on GIRK2 activation was tested. Of the four variant analogs synthesized, GiGA1 showed high specificity for activating GIRK1/GIRK2 channels.
The predicted binding mode of GiGA1 to a model of the alcohol pocket revealed that the O-methyl phenyl moiety of GiGA1 formed π-π interactions with phenylalanine (F) and Leucine (L) present on GIRK1. This revealed the superior binding of GiGA1 relative to the additional analogs whose extra methyl group present a potential clashing with the π-forming amino acids. This event renders them less optimal in binding.
Mechanism of GiGA1 action on potassium channels
Mutation studies did not reveal the mechanism of specificity; this led to the hypothesis that GiGA1 specificity is dependent on additional structural features.
Taken together, Zhao et al. demonstrate that GiGA1 is a GIRK1-specific modulator that allosterically binds at the alcohol binding pocket. Additional structural studies are necessary to determine the precise binding mechanism.
GiGA1 presents a greater advantage over alcohol in its greater potency, ability to saturate, and rapid deactivation rate. The latter offers the potential for acute adjustment in channel activity.
The potential of potassium channel activator in other neurological disorders
GIRK channels are instrumental in controlling excitability and preventing seizures. Augmenting the GIRK1/2 function, therefore, induces a protective effect. GiGA1 action also occurs at the hippocampal pyramidal neurons alongside the hippocampal pyramidal neurons found in the brain at large.
This additional effect is supplementary to seizure prevention as the hippocampus plays an essential role in the occurrence and propagation of epilepsy. Following this knowledge, GiGA1 reduced both the frequency and severity f seizures; an effect comparable to a commonly used antiepileptic drug.
Some side effects include dose-dependent sedation, suggesting future optimization of GiGA1 is necessary to lessen this effect.
The group suggests that the property of selective activation of GIRK1/GIRK2 will be useful in the treatment of brain disorders, notably alcohol use disorder (AUD). Combined with the known role of the hippocampus in alcohol addiction and its predominant GIRK1/2 composition, GiGA1 was cited as a molecule for the prevention of neuronal plasticity associated with AUD.
GiGA1 may additionally offer non-opioid therapy for producing analgesia owing to the implication of GIRK channels in pain perception.
Zhao et al. conclude that “pharmacologically targeting subsets of GIRK channels in the brain may afford new opportunities for developing advanced therapeutics for treating various human neurological disorders”.
- Zhao, Y. et al. (2020) Identification of a G-Protein-Independent Activator of GIRK Channels. Cell Reports. DOI: doi.org/10.1016/j.celrep.2020.107770.