Article pubs.acs.org/jnp
HPLC-Based Activity Profiling for GABAA Receptor Modulators in Extracts: Validation of an Approach Utilizing a Larval Zebrafish Locomotor Assay Fahimeh Moradi-Afrapoli,† Samad Nejad Ebrahimi,†,‡ Martin Smiesko,§ and Matthias Hamburger*,† †
Pharmaceutical Biology and §Division of Molecular Modeling, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland ‡ Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran S Supporting Information *
ABSTRACT: Gamma-aminobutyric acid type A (GABAA) receptors are major inhibitory neurotransmitter receptors in the central nervous system and a target for numerous clinically important drugs used to treat anxiety, insomnia, and epilepsy. A series of allosteric GABAA receptor agonists was identified previously with the aid of HPLC-based activity profiling, whereby activity was tracked with an electrophysiological assay in Xenopus laevis oocytes. To accelerate the discovery process, an approach has been established for HPLC-based profiling using a larval zebrafish (Danio rerio) seizure model induced by pentylenetetrazol (PTZ), a pro-convulsant GABAA receptor antagonist. The assay was validated with the aid of representative GABAergic plant compounds and extracts. Various parameters that are relevant for the quality of results obtained, including PTZ concentration, the number of larvae, the incubation time, and the data analysis protocol, were optimized. The assay was then translated into an HPLC profiling protocol, and active compounds were tracked in extracts of Valeriana off icinalis and Magnolia off icinalis. For selected compounds the effects in the zebrafish larvae model were compared with data from in silico blood−brain barrier (BBB) permeability predictions, to validate the use for discovery of BBB-permeable natural products.
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thereby supporting the validity of natural products directed efforts in the search for novel GABAA receptor modulators. Various assay formats have been used for the discovery of GABAA receptor ligands, such as radioimmunoassays and fluorescent labels to identify compounds interacting at the benzodiazepine binding site,13,14 while radioactivity-based ion flux assays,15 microphysiometry,16 and the use of fluorescent dyes that are sensitive to chloride ions have been used to examine ion channel function.17,18 Patch clamp and twomicroelectrode electrophysiological assays have been employed with Xenopus laevis oocytes and HEK cells in which GABAA receptors were transiently or stably expressed.19,20 A semiautomated assay with Xenopus oocytes has been successfully used for the screening of extract libraries, HPLC activity profiling for hits, and subsequent structural optimization of active hits.8,9,11,12,20−32 In HPLC-based activity profiling of extracts, the turnaround time of bioassay data is the limiting step.33,34 To accelerate the discovery and dereplication process, we have established and validated in the present work an in-house assay utilizing a
amma-aminobutyric acid type A (GABAA) receptors are the key inhibitory neurotransmitter receptors in the central nervous system (CNS). They are heteropentamers composed of different subunits forming a central pore permeable to chloride and bicarbonate ions. The most abundant GABAA receptor subtype in the mammalian CNS is an assembly of two α1, two β2, and one γ2 subunit.1 GABAA receptors are targets for numerous clinically important drugs used to treat anxiety, panic, insomnia, and epilepsy.2,3 However, current drugs such as the benzodiazepines and nonbenzodiazepines (the so-called Z-drugs) are associated with side-effects due to a lack of receptor-subtype selectivity.2 Also, the scaffold diversity of synthetic drugs and experimental compounds targeting GABAA receptors is very limited. The need for the discovery of allosteric GABAA receptor modulators with novel scaffolds that possibly target binding sites other than the benzodiazepine binding site is thus apparent. Various natural products with scaffolds that are new for GABAA receptors have been reported in recent years,4−8 and compounds such as piperine have been shown to interact at a benzodiazepineindependent allosteric binding site.9 Medicinal chemistry efforts starting from piperine led to in vivo active analogues with improved selectivity and biopharmaceutical properties,10−12 © 2017 American Chemical Society and American Society of Pharmacognosy
Received: January 26, 2017 Published: May 9, 2017 1548
DOI: 10.1021/acs.jnatprod.7b00081 J. Nat. Prod. 2017, 80, 1548−1557
Journal of Natural Products
Article
behavioral model with zebrafish larvae. In recent years, zebrafish (Danio rerio) has become an increasingly important model organism in drug discovery,35−38 being ranked by the National Institutes of Health (NIH) as the third most important experimental organism after rats and mice.39 It has a high genetic homology (10 mM) were also included (Figure 1A). Patterns of larval response to different PTZ concentrations were visualized by plotting movement vs time, whereby total distance traveled in 5 min intervals was summed (Figure 1B). Locomotor activity increased following exposure to 5 mM PTZ and remained constant within 30 min. The movement induced by 10 mM PTZ gradually increased within 15 min due to the increasing frequency of whirlpool-like movements (stage II) 1549
DOI: 10.1021/acs.jnatprod.7b00081 J. Nat. Prod. 2017, 80, 1548−1557
Journal of Natural Products
Article
compound was assessed prior to screening for activity. Signs of toxicity were monitored between 1.5 and 24 h of incubation. The toxicity rate (total number of impaired and dead larvae) did not change significantly between 3 and 24 h (data not shown). It was thus assumed that an exposure of 3 h was sufficient for the compounds to be absorbed and to exhibit toxicity and/or activity. The maximal tolerated concentration (MTC) of each compound, as determined in the toxicity assessment (Table 1), was then used as the highest test concentration. Lower test concentrations were prepared by serial dilutions. Table 1. Maximum Tolerated Concentrations (MTC) of Pure Compounds and Plant Extracts compound
MTC (μM)
diazepam
20
magnolol (1) valerenic acid (2) piperine (3)
4 10 12
compound
MTC (μM)
sophoraflavanone G (4) sanggenon C (5)
10
dehydroabietic acid (6) sandaracopimaric acid (7)
6
2
extract M. off icinalis V. off icinalis
MTC (μg/ mL) 4 12
2
Benzodiazepines are the most frequently prescribed GABAA receptor modulators and are highly effective inhibitors of PTZevoked seizures in rodents.47 Therefore, the assay protocol was evaluated first with diazepam (Figure 2). Diazepam (MTC 20 μM) lowered PTZ-provoked locomotor activity in zebrafish larvae in a concentration-dependent manner at concentrations of
