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  • br Introduction The generic antifibrinolytic drug tranexamic


    Introduction The generic antifibrinolytic drug tranexamic PU-WS13 (TXA) inhibits the protein-protein interaction between plasminogen and fibrin [9]. TXA is frequently used to reduce bleeding in various hemorrhagic conditions, such as during surgical procedures in the heart [19]. However, treatment with TXA is associated with an increased incidence of seizures [15], [19]. These adverse events are hypothesized to be due to the competitive antagonist properties of TXA towards the inhibitory neuronal receptors for GABAA and, possibly, glycine [7], [12], [15]. A recent search for novel PBIs identified 4-PIOL with improved potency for the primary target, but this compound still shows insufficient selectivity towards the inhibitory GABAA receptor [3]. In this effort the selectivity against the GABAA receptor was measured using an animal derived low-throughput membrane binding assay. This counter-screening assay was accurate, but labor intensive, and is thus not considered suitable for assessments across larger compound sets. In addition, this assay provides no information on agonist or antagonist mode of action. After similar internal assay experiences during the identification of candidate drug AZD6564 [4], we aimed to derive a more efficient and informative screening assay for the GABAA receptor, a very important CNS target both for clinical efficacy and toxicology. In short, the aim was to establish an assay which allows for the identification of activity towards inhibitory neuron receptors GABAA and glycine in a high-throughput screening setting. A prerequisite for these activities was to simultaneously improve translatability through a move from animal-derived cells towards more primary cell types of human origin. Human induced pluripotent stem cells (hiPSC) provide large scale availability of human cells and can potentially be used to address the very limited accessibility of human primary neurons. Human iPSC derived neurons express endogenous levels of inhibitory neuronal receptors [5], [21], [22] and have been shown to recapitulate important electrophysiological features of neurons using calcium imaging [5], multi-electrode systems (MEA) [1] and patch clamping [8], [22]. However, both manual and automated patch clamping [6] are challenging to perform in a screen setting, while MEA is associated with significant analytical and computational challenges [17]. Additionally, neither method is suitable and sufficiently cost effective to allow for high-throughput screens in their current formats. Instead we turned our interest to label free dynamic PU-WS13 mass redistribution (DMR) technology, which measures the integrative phenotypic response originating from multiple signaling events in the cells [11]. The technology allows for the use of high density microplates with low cell numbers and short turn-around time making it a suitable candidate for our purpose. In this study we develop and validate the use of human iPSC derived neurons for measurements of endogenous receptor modulation using high-throughput DMR technology. We show that the inhibitory GABAA and glycine receptors can be robustly modulated with natural ligands. Moreover, we can detect agonist selectivity between receptor populations comprised of different subunit compositions [18], allosteric modulator potentiation [10], [22], and taurine dual receptor agonism [2], thus providing confidence in assay applicability for mechanistic studies. We further demonstrate that our method can detect previously reported off-target activity of TXA against the GABAA and glycine receptors. Additionally, we demonstrate proof of concept for the evaluation of compounds from a lead series of PBIs for GABAA receptor with improved selectivity profile. Our study demonstrates the direct applicability and impact of using label free high-throughput technology with human iPSC neurons in drug discovery efforts.
    Materials and methods
    Discussion In this study we applied hiPSC neurons in a label-free dynamic mass redistribution assay in high-throughput format. Previous publications using hiPSC neurons from the same supplier demonstrate EC50 values of 0.43 and 0.25 μM for GABA using patch clamping [22] and calcium imaging [5], respectively. This is in excellent agreement with our EC50 value of 0.33 μM from the DMR assay. Earlier studies based on DMR-technology has shown higher EC50 values for both GABA at 2.9 μM and gabazine at 7.4 μM using IMR32 cells [11]. The 10-fold difference compared to our data could be due to cell model differences. We further show that at the EC20 for GABA (90 nM) diazepam acts as a positive allosteric modulator and can potentiate the GABAA receptor by shifting the CRC to the left. This confirms previous results in hiPSC neurons [10], [22]. Additionally, in agreement with our own data, mice brain slices exposed to taurine at high concentrations (3 mM) has been shown to activate both GABAA and glycine receptors, whereas lower concentrations (0.5 mM) only activates the glycine receptor [2]. Similar findings are also described for rat CA1 neurons [20]. Finally, a study using patch clamping in hiPSC shows an EC50 of glycine stimulation at 0.17 mM [10], compared to 11 μM in our study. The difference could be explained by differences in protocol for hiPSC neuron derivation. Based on these data we conclude that we can replace a previous rat based low-throughput assay [3], [4] with a high-throughput assay based on human cells and in doing so additionally provide information on the compound mode of action.