Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 Evodia preparations.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines, In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study.

A neolignan from Connarus tuberosus as an allosteric GABAA receptor modulator at the neurosteroid binding site.

In a screening of a small library of extracts from plants of the Amazonian and Cerrado biomes, a hexane extract of Connarus tuberosus roots was found to significantly potentiate the GABA induced fluorescence in a fluorescence (FLIPR) assay in CHO cells stably expressing the α1ß2γ2 subtype of human GABAA receptors. With the aid of HPLC-based activity profiling the activity was linked to the neolignan connarin. In CHO cells the activity of connarin was not abolished by increasing concentrations of flumazenil, while the effect of diazepam was increased by increasing concentrations of connarin. The effect of connarin was abolished by pregnenolone sulfate (PREGS) in a concentration-dependent manner, and the effect of allopregnanolone was further increased by increasing concentrations of connarin. In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes transiently expressing GABAA receptors composed of human α1ß2γ2S and α1ß2 subunits connarin potentiated the GABA-induced currents, with EC50 values of 1.2 ± 0.3 µM (α1ß2γ2S) and 1.3 ± 0.4 µM (α1ß2), and with a maximum enhancement of currents Emax of 1959 ± 70% (α1ß2γ2S) and 185 ± 48% (α1ß2). The activation induced by connarin was abolished by increasing concentrations of PREGS.

Apo2ph4: A Versatile Workflow for the Generation of Receptor-based Pharmacophore Models for Virtual Screening.

Pharmacophore models are widely used as efficient virtual screening (VS) filters for the target-directed enrichment of large compound libraries. However, the generation of pharmacophore models that have the power to discriminate between active and inactive molecules traditionally requires structural information about ligand-target complexes or at the very least knowledge of one active ligand. The fact that the discovery of the first known active ligand of a newly investigated target represents a major hurdle at the beginning of every drug discovery project underscores the need for methods that are able to derive high-quality pharmacophore models even without the prior knowledge of any active ligand structures. In this work, we introduce a novel workflow, called apo2ph4, that enables the rapid derivation of pharmacophore models solely from the three-dimensional structure of the target receptor. The utility of this workflow is demonstrated retrospectively for the generation of a pharmacophore model for the M2 muscarinic acetylcholine receptor. Furthermore, in order to show the general applicability of apo2ph4, the workflow was employed for all 15 targets of the recently published LIT-PCBA dataset. Pharmacophore-based VS runs using the apo2ph4-derived models achieved a significant enrichment of actives for 13 targets. In the last presented example, a pharmacophore model derived from the etomidate site of the α1ß2γ2 GABAA receptor was used in VS campaigns. Subsequent in vitro testing of selected hits revealed that 19 out of 20 (95%) tested compounds were able to significantly enhance GABA currents, which impressively demonstrates the applicability of apo2ph4 for real-world drug design projects.

ß subunits of GABAA receptors form proton-gated chloride channels: Insights into the molecular basis.

Gamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of ß-subunit homomers as proton-gated anion channels. Mutation of a single H267A in ß3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the ß3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.

Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators.

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABAA receptors with an α1ß2γ2 subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC50 values of 0.5, 4.6, and 1.4 µM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABAA receptors of the α1ß2γ2S and α1ß2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC50 (α1ß2γ2S) = 43.6 µM; Emax = 809% and EC50 (α1ß2) = 57.6 µM; Emax = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

Tetrapeptide Ac-HAEE-NH2 Protects α4ß2 nAChR from Inhibition by Aß.

The cholinergic deficit in Alzheimer's disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aß peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the 11EVHH14 site in Aß peptide mediates its interaction with α4ß2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aß-α4ß2 nAChR complex is based on the interaction of 11EVHH14 with its charge-complementary counterpart in α4ß2 nAChR. Indeed, we discovered a 35HAEE38 site in α4ß2 nAChR, which is charge-complementary to 11EVHH14, and molecular modeling showed that a stable Aß42-α4ß2 nAChR complex could be formed via the 11EVHH14:35HAEE38 interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH2 can bind to Aß via 11EVHH14 site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH2 tetrapeptide completely abolishes the Aß42-induced inhibition of α4ß2 nAChR. Thus, we suggest that 35HAEE38 is a potential binding site for Aß on α4ß2 nAChR and Ac-HAEE-NH2 tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4ß2 nAChR-dependent cholinergic dysfunction in AD.

From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs.

The proteins of the Ly6 family have a three-finger folding as snake venom α-neurotoxins, targeting nicotinic acetylcholine receptors (nAChRs), and some of them, like mammalian secreted Ly6/uPAR protein (SLURP1) and membrane-attached Ly-6/neurotoxin (Lynx1), also interact with distinct nAChR subtypes. We believed that synthetic fragments of these endogenous proteins might open new ways for drug design because nAChRs are well-known targets for developing analgesics and drugs against neurodegenerative diseases. Since interaction with nAChRs was earlier shown for synthetic fragments of the α-neurotoxin central loop II, we synthesized a 15-membered fragment of human Lynx1, its form with two Cys residues added at the N- and C-termini and forming a disulfide, as well as similar forms of human SLURP1, SLURP2, and of Drosophila sleepless protein (SSS). The IC50 values measured in competition with radioiodinated α-bungarotoxin for binding to the membrane-bound Torpedo californica nAChR were 4.9 and 7.4 µM for Lynx1 and SSS fragments, but over 300 µM for SLURP1 or SLURP2 fragments. The affinity of these compounds for the α7 nAChR in the rat pituitary tumor-derived cell line GH4C1 was different: 13.1 and 147 µM for SSS and Lynx1 fragments, respectively. In competition for the ligand-binding domain of the α9 nAChR subunit, SSS and Lynx1 fragments had IC50 values of about 40 µM, which correlates with the value found for the latter with the rat α9α10 nAChR expressed in the Xenopus oocytes. Thus, the activity of these synthetic peptides against muscle-type and α9α10 nAChRs indicates that they may be useful in design of novel myorelaxants and analgesics.