Written by Marc Rogers PhD and Sophie Rose PhD
Metrion’s first “Ion channel drug discovery” webinar
Metrion Biosciences’ inaugural ion channel webinar took place at the end of June and featured a presentation by Dr Jon Lippiat, from the School of Biomedical Sciences at the University of Leeds. Jon presented an inspiring and thought-provoking talk focusing on his recent work which involved using structure-based virtual screening techniques to identify novel inhibitors of the hKNa1.1 channel. Prior knowledge of the structure of the channel, including insight into the potential binding domains had been gained using cryo-electron microscopy. Attendees gathered from across the globe and there was a brief introduction prior to Jon’s presentation and a spirited question and answer session afterwards, both presented by Metrion’s CSO Dr Marc Rogers.
The importance of the KNa1.1 channel and the journey to find novel inhibitors
Jon focused his presentation on the KNa1.1 ion channel, which is encoded by the KCNT1 gene and is one of the 4 SLO-related, RCK-domain containing human potassium channels. The channel is tetrameric and comprised of six transmembrane segments. Jon’s group have been studying the epileptic disorders associated with mutations of the KCNT1 gene, which unfortunately are not well controlled by anti-epileptic drugs. Sadly, many children suffering from these symptoms are severely affected and their life expectancy is reduced so there is a high unmet clinical need. Generally, all mutations are heterozygous and dominant with mutations resulting in gain-of-function. Prior to the year 2020, the only known inhibitors of this channel were Quinidine, Bepridil and Clofilium, all of which are rather non-specific, lack potency, and are associated with significant side-effects. The only inhibitor trialled in clinical use is quinidine, in an attempt to suppress over-active channels in these patients. However, due to its effects on ion channels in the heart, quinidine dosing is very limited. To understand how inhibitors interact with the human KNa1.1 channel and identify new inhibitors, the published structures of the chicken KNa1.1 homologue have been used for virtual docking and mutagenesis studies.
Uncovering the mutation required for channel inhibition
KNa1.1 can be studied using whole-cell recordings from HEK293 cells transiently transfected with wild-type or mutant human KNa1.1 channel proteins. Channel activation is weakly voltage-dependent and slowly time dependent compared with other potassium channels. It was clear from Jon’s work that phenylalanine residue 346 (F346) is required for KNa1.1 inhibition by both Quinidine and Bepridil. A disease-causing mutation (Phe to Leu switch) has since been published. Mutations of F346 cause an increase in the IC50 to at least ten-fold higher concentrations with both Quinidine and Bepridil, in line with the docking studies.
Screening of a large virtual compound library
To further probe the structure of the active pore domain, Jon then discussed how a virtual library of 100,000 compounds was screened to give predictions of free energy changes and suggest which drug compounds may induce channel inhibition. They supplemented this with a ligand-based approach whereby compounds similar in structure to Bepridil were selected and purchased for further functional validation. They completed functional analysis of 17 compounds at 10mM against the wild-type channel, using manual patch clamp electrophysiology whole cell recordings.
The active compounds were counter-screened against the channel carrying the core F346 mutation and evaluated in concentration inhibition experiments. IC50 values versus the wild-type channel and the Y796H disease mutation were plotted and compared to the data observed for both Quinidine and Bepridil. 6 of the 17 compounds showed inhibition of the human KNa1.1 channel at 10mM, and so additional compounds with similar structures were purchased and tested. Small changes to the basic structure resulted in a loss of inhibition of the channel. This demonstrates the specificity of the docking approach and the significant involvement of the inner side of the channel pore. Terminal trifluoromethyl groups are a common feature of the compounds in their virtual screens and this motif is known to position itself within the pore of the channel.
Preliminary safety/ toxicology findings
This structure-based approach delivered compounds with potential to inhibit other potassium channels, such as hERG. This potential cardiac safety risk was probed in more detail and it was found that only three of these compounds exhibited strong hERG inhibition, demonstrating good potential for developing potent, safe and selective KNa1.1 modulators. Jon’s group also investigated cytotoxicity using HEK cells incubated overnight in a cell viability assay, using positive controls 10% DMSO and Blasticidin. The selected compounds showed very little cytotoxic effect across the concentration range, increasing their potential as starting points for further drug discovery efforts.
KNa1.1 channel inhibitors
Jon then discussed further KNa1.1 channel inhibitors which have been published. He highlighted studies published by a group at Vanderbilt University (this involved a thallium flux fluorescent HTS assay) and by Praxis Precision Medicine (Cambridge, MA). Praxis employed high throughput rubidium flux and atomic absorption spectroscopy to identify hits and develop their lead compound. These compounds appear to have efficacy in reducing rodent CNS epileptic activity in vitro and in vivo.
The structures of the various KNa1.1 channel inhibitors described in the presentation were given, which have been identified and published over the past year and a half. Compounds denoted “BC5”, “BC6” and “BC7” came through the ligand-based approach and are structurally similar to Bepridil, but strongly inhibit hERG channels. The frequent appearance of the trifluoromethyl group in docked compounds remains important and two of these compounds displayed this motif. One of the compounds was particularly potent at inhibiting wild-type channels, but less so for those containing disease causing GoF mutations.
Progressing structure-based approaches
Having demonstrated that structure-based virtual screening can deliver novel inhibitors of the hKNa1.1 channel, this work has been expanded to another virtual screen using a 9 million compound ZINC library on the High-Performance Computing ARC3 system located at the University of Leeds. This adds an extra layer to the workflow and enables the screening of much larger virtual compound libraries and the further characterisation of additional and novel hit compounds. The group have also developed a concatemeric construct KNa1.1 WT-T2A-Y796H and a stable cell line which will be used in medium throughput fluorescence and automated patch clamp experiments. Jon’s colleagues have also been generating their own ion channels structures at the University of Leeds using cryo-electron microscopy to determine how drugs interact with a number of different ion channels implicated in human disease.
Jon concluded that high resolution cryo-EM structures of KNa1.1 can be used both to model inhibitor binding and in virtual HTS. There is a high degree of homology between species and strongly conserved domains. This enables the use of non-human protein structures. The potential success of this approach is that a small-scale virtual screen can be evaluated initially using libraries of reasonably priced and readily available screening compounds to ascertain the hit rate before embarking upon a larger functional evaluation. The limitation here however is that knowledge is required of a viable binding site. To escape potential cross reactivity issues, it would be favourable to target other parts of the protein structure, such as sodium binding sites. The study has resulted in a selection of tool compounds for further characterisation and to probe channel function, and Jon’s group hope to be able to address the high unmet clinical need associated with KCNT1 associated epileptic disorders using this combined approach. The project was supported by the BBSRC, UKRI, Autifony Therapeutics and Action Medical Research for Children.
The full presentation including the Q&A session is available to all registrants. If you did not register for the event but are interested in accessing the presentation please email: firstname.lastname@example.org.