Discovery and ADME profiling of CNS-active natural products

Gamma aminobutyric acid type A (GABAA) receptors are the major inhibitory neurotransmitter receptors in the Central Nervous System (CNS). Several clinically important drugs used to treat anxiety, insomnia and epilepsy act via an allosteric modulation of postsynaptic GABAA receptors. The currently used drugs are associated with serious side-effects, mainly due to a lack of receptor subtype selectivity. This raises the medical need for discovery of novel types of GABAA receptor modulators. We previously identified a series of allosteric GABAA receptor agonists with the aid of HPLC-based activity profiling, whereby activity was tracked with an electrophysiological assay in Xenopus oocytes expressing GABAA receptors of desired subunit composition. In an expansion of our investigations, aiming at acceleration of the activity profiling, an in-house assay was established using larval zebrafish locomotor activity model. In that, larval convulsions were provoked by the pro-convulsant GABAA receptor antagonist pentylenetetrazol (PTZ), and GABAA receptor agonistic extracts and compounds were identified through a decrease in larval locomotion. The assay was validated with the aid of known GABAergic compounds that had previously shown activity in the Xenopus oocyte assay. Assay validation was approached with respect to parameters relevant for the quality of results, including PTZ concentration, number of larvae, concentration of test samples, duration of incubation with test solutions and tracking of larval locomotion, as well as data visualization protocol. The validated protocol was subsequently translated into an HPLC-based activity profiling protocol using ethyl acetate extracts of Valeriana officinalis and Magnolia officinalis. The zebrafish larvae locomotor assay was later employed for activity profiling of South African medicinal plants traditionally used for the treatment of epilepsy and other neurological disorders. An initial screening of medicinal plants in Xenopus oocytes patch clamp assay revealed GABAergic activity of a dichloromethane extract from leaves of Searsia pyroides. The extract significantly lowered PTZ-provoked locomotion in zebrafish larvae when tested at 4 μg/mL. HPLC-based profiling followed by targeted isolation of phytochemicals in the active timewindow, revealed identification of three anacardic acid derivatives (1-3). Also, three structurally related compounds (4-6) were purified from inactive areas. After assessment of the isolated compounds with both Xenopus oocyte and larval zebrafish models, GABAA receptor modulation activity of the extract was successfully correlated with the phytochemicals in the active time-window. Additionally, in the zebrafish larval assay a series of GABAA receptor agonistic natural products, previously identified by the Xenopus oocyte assay, were tested. Lowering of locomotor activity was found for these compounds with exception of sanggenone C. Physicochemical and biochemical properties (PSA, cLogP, number of H-donor and acceptor sites, and number of rotatable bonds) of tested compounds were calculated in silico. A lack of permeability across the blood-brain-barrier (BBB) was concluded for sanggenone C, inferring its lack of activity in vivo, which also remarked the exclusivity of the zebrafish larvae assay for discovery of BBB permeable natural products. The last part of our research focused on the membrane permeability of kaempferol (KMF), a sedative flavonoid which targets GABAA receptors, and its major intestinal metabolite, 4-hydroxyphenylacetic acid (4-HPAA). In previous studies, KMF induced sedative effects in mice only after oral administration (i.o.) but not after intraperitoneal application (i.p.). However, 4- HPAA, the biotransformation product of KMF by intestinal microflora, induced behavioral changes after i.p. injection. To further explore the relation between KMF and 4-HPAA bioactivities and their route of administration, their ability to cross biological barriers has been examined. Intestinal barrier permeability studies were performed with Caco-2 cells, and bloodbrain barrier transport studies were done with an immortalized mono-culture human model and a primary triple-co-culture rat model. UHPLC–MS/MS quantification methods for bioanalysis of KMF and 4-HPAA in the corresponding transport media were developed and validated according to international guidelines. The fundamental validation parameters included accuracy, precision, specificity, selectivity, sensitivity, repeatability, reproducibility, short-term and long-term stabilities. Data obtained with all barrier models were indicative of high intestinal and BBB permeation of KMF, and no permeation for 4-HPAA when compared with the fluorescent barrier integrity markers. In all experiments efflux ratios were below two, indicating that no active transport processes were involved for both compounds. Within a calcein-AM uptake assay in porcine brain capillary endothelial cells, kaempferol and 4-HPAA were found neither Pglycoprotein substrates nor P-glycoprotein inhibitors. Our in vitro data supported the previous described in vivo CNS effects of KMF while the role of 4-HPAA needs to be further studied

HPLC-Based Activity Profiling for GABAA Receptor Modulators in Extracts: Validation of an Approach Utilizing a Larval Zebrafish Locomotor Assay

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 officinalis and Magnolia officinalis. 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

Cinnamoylphenethyl Amides from Polygonum hyrcanicum Possess Anti-Trypanosomal Activity

A methanolic extract from aerial parts of Polygonum hyrcanicum (Polygonaceae) showed high activity against Trypanosoma brucei rhodesiense (IC50 = 3.7 microg/mL). Bioassay-guided fractionation of the extract resulted in isolation of cinnamoylphenethyl amides, including N-trans-caffeoyltyramine (1), N-trans-p-coumaroyltyramine (7), and N-trans-feruloyltyramine (8) as the main active constituents (IC50s ranging from 2.2 to 13.3 microM). Some structurally related, but less active compounds, such as cannabisin B (2), tyrosol (3), p-coumaric acid (4), ferulic acid (5), and N-cis-feruloyltyramine (6) were also identified, along with N-trans-3,4-dimethoxycinnamoyldopamine (9). Cytotoxicity of the active compounds in L6 cells was determined, and selectivity indices (SI) of 7.9 to 33.4 were calculate

HPLC-based activity profiling for GABAA receptor modulators from Murraya exotica

A dichloromethane extract from twigs and leaves of Murraya exotica produced allosteric potentiation of gamma aminobutyric acid (GABA) induced chloride currents in a microelectrode assay in Xenopus laevis oocytes expressing GABA receptors of α1, β2, γ2s subunit composition. The activity was tracked by HPLC-based activity profiling utilizing a zebrafish locomotor activity assay. Osthol (9) was identified as the main active compound. In addition, five other coumarins and four flavonols were identified. Osthol (9) and structurally related coumurrayin (10) were tested in the Xenopus oocyte assay. Compound 9 potentiated GABAA-induced chloride currents by 487 ± 42%, with an EC50 of 46 ± 10 μM, while 10 showed negligible effects on chloride currents. In silico evaluation of physicochemical properties showed that 9 and 10 had properties that are favorable for oral bioavailability and BBB permeability

Evaluation of the Cytotoxicity of Satureja spicigera and Its Main Compounds

Satureja spicigera (Lamiaceae) grows wildly in Northwest of Iran. In this study, bioassay-guided isolation and identification of the main compounds has been reported using various chromatographic methods and comparison of their spectral data with those reported in the literature. Brine shrimp lethality and four cancerous cell lines HT29/219, Caco2, NIH-3T3, and T47D were used for cytotoxicity evaluations. From the aerial parts of S. spicigera, nine known compounds including two flavanones, 5,7,3,5-tetrahydroxy flavanone (8) and 5,4-dihydroxy-3-methoxyflavanone-7-(6-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (9), one dihydrochalcone, nubigenol (7), together with thymoquinone (1), thymol (2), carvacrol (3), β-sitosterol (4), ursolic acid (5) and oleanolic acid (6) were identified. Among the isolated chalcone and flavanones, compound 8 was effective against Artemia salina larva (LC50= 2 μg/mL) and only the compound 9 demonstrated IC50 value of 98.7 μg/mL on the T47D (human, breast, ductal carcinoma). Other compounds did not show significant inhibition of the cell growth

Bisabololoxide derivatives from Artemisia persica, and determination of their absolute configurations by ECD

Five antiplasmodial bisabololoxide sesquiterpene diesters were isolated from an EtOAc extract of the aerial parts of Artemisia persica following an HPLC-time-based activity profiling of the extract. Structure elucidation was achieved by 1D and 2D NMR experiments. Relative configurations of cyclohexenone/cyclohexene and tetrahydropyran moieties of 1-5 were established on the basis of (3)J(H-H) coupling constants and NOE difference spectra. Stereochemical correlation of the two rings, and assignment of absolute configuration of 1-5 were achieved by comparison of experimental ECD spectra with simulated ECD data for possible stereoisomers, by using time dependent density function theory (TDDFT). Bisaboloids 1-4 exhibited in vitro antimalarial activity against Plasmodium falciparum, with IC(50) values ranging from 2.8 to 20.1μM, and selectivity indices (SI) in L-6 cells of 3.7-11.9

Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro BBB and intestinal drug permeability studies

Sedative and anxiolytic-like properties of flavonoids such as kaempferol and quercetin, and of some of their intestinal metabolites, have been demonstrated in pharmacological studies. However, routes of administration were shown to be critical for observing in vivo activity. Therefore, the ability to cross intestinal and blood-brain barriers was assessed in cell-based models for kaempferol (KMF), and for the major intestinal metabolite of KMF, 4-hydroxyphenylacetic acid (4-HPAA). Intestinal transport studies were performed with Caco-2 cells, and blood-brain barrier transport studies with an immortalized monoculture human model and a primary triple-co-culture rat model. UHPLC–MS/MS methods for KMF and 4-HPAA in Ringer-HEPES buffer and in Hank’s balanced salt solution were validated according to industry guidelines. For all methods, calibration curves were fitted by least-squares quadratic regression with 1/X2 as weighing factor, and mean coefficients of determination (R2) were >0.99. Data obtained with all barrier models showed high intestinal and blood-brain barrier permeation of KMF, and no permeability of 4-HPAA, when compared to barrier integrity markers