Profiling for Natural Products with GABAA Receptor Modulatory and Immunosuppressant Activities

HPLC-based activity profiling is an effective approach for tracking active compounds in an extract. In this doctoral thesis, this approach was applied to discover new scaffolds for Gamma-aminobutyric acid type A (GABAA) receptor modulators and immunosuppressant agents from natural origin.
GABAA receptors are the major inhibitory neurotransmitter in the human brain. They are also a therapeutic target in drug discovery. Lack of subtype-selectivity of GABAergic drugs correlates with undesireable side-effects. Benzodiazepines, barbiturates, neurosteroids, anesthetics, and alcohol are well-known modulators of GABAA that bind to its receptor. Some methodological approaches have been developed for investigating GABAA receptor modulator pharmacology and functional response. The gold standard for the functional assay of GABA is in vitro Xenopus oocyte assay, which is expressing the α1β2γ2s subunit compositions. This method was applied as primary screening for the in vivo in house assay utilizing zebrafish larvae in this research.
The HPLC-based activity profiling and screening using zebrafish larvae locomotor assay allowed us to identify active compounds in dichloromethane (DCM) extract of twigs and leaves of Murraya exotica L. Ten compounds were isolated from DCM extract consisting of six coumarins and four polymethoxyflavones. The active compound was identified as osthol (9). It potentiated GABAA-induced chloride currents by 487 ± 42%, with an EC50 of 46 ± 10 μM in Xenopus oocyte assay. The adjacent compound in the activity profiling showed a related structure (coumurrayin (10)) with an additional methoxy group, but it showed a neglectable effect. Furthermore, the physicochemical properties of these two compounds were calculated in silico. The result shows that both compounds are favourable for oral bioavailability and blood-brain barrier (BBB) permeability.
A library of 798 ethyl acetate (EtOAc) extracts was screened in fluorometric imaging plate reader (FLIPR) assay. This functional assay utilized Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α1β2γ2 subunit composition. The active extracts were further checked for concentration-dependent effects. Afterwards, the active extracts were submitted to HPLC-based activity profiling to localize the active compounds in the extracts. This screening resulted in two active extracts, which one of them was an EtOAc extract from Casearia corymbosa Kunth leaves. Eight compounds were isolated from active time windows, and one compound was obtained from an adjacent fraction. All of the compounds were clerodane-type diterpenoids, of which five were new. All compounds were screened in FLIPR assay. Five compounds (1, 3, 5, 7, 8) exhibited activation for allosteric GABAA receptor modulators and were further assessed for concentration-response and determination of EC50 values. Compounds 3, 7, and 8 displayed significantly enhanced potentiation of GABA with EC50’s of 0.51, 4.57, and 1.36 μM, respectively. In contrast, compounds 1 and 5 did not show concentration-dependent effect.
Compound 8 was further evaluated to identify allosteric GABAA receptor modulatory binding sites in FLIPR assay. In order to evaluate the affinity to the benzodiazepines binding site, compound 8 (5 µM) combined with increasing concentrations of flumazenil and increasing concentrations of compound 8 combined with diazepam (2 µM) were tested. For the barbiturates binding site, increasing concentrations of compound 8, combined with etazolate (0.78 µM) were assessed. For the neurosteroids binding site, the same treatments as for benzodiazepine binding site assessments were used consisting of compound 8 with increasing concentrations of pregnenolone sulfate (PREGS) and increasing concentrations of 8 with a fixed concentration of allopregnanolone at 0.25 µM. The results showed that additive potentiation could be observed in the addition of diazepam, etazolate, and allopregnanolone. However, decreasing activation was observed with the addition of compound 8 at the highest concentration of PREGS. This suggests that compound 8 and PREGS bind at the same sites on the neurosteroid GABAA receptors with compound 8 being the first reported non-steroidal compound interacting at the neurosteroid binding site.
Toxicity and side effect issues in immunosuppressant drugs were the driving force in searching for a natural product as a source of immunosuppressive agents. A library of 435 extracts from traditional Chinese herbal medicines was screened for inhibition of human T lymphocyte proliferation. One of the active extracts without concomitant toxicity, a DCM extract from the roots of Scutellaria baicalensis, was submitted to HPLC-based activity profiling for tracking the active compounds. Seventeen flavonoids were isolated in the time active windows and its adjacent fractions. The isolated flavonoids possessed unusual substitution patterns in B-ring, with preferred substitutions at C-1’ and C-6’ and lack of substitutions at C-3’ and C-4’. All active compounds showed cytotoxicity at a concentration of 100 µg/mL. Compounds 2, 4, 8, and 12 displayed moderate activity with EC50 values of 55.9, 45.9, 61.6, and 50.2 µM, respectively. High immunosuppressant activity was exhibited by compounds wogonin (11), chrysin (13), skullcapflavone (14), and oroxylin A (16) with EC50 values of 20.2, 22.2, 12.2, and 39.0 µM, respectively.
All structures of the isolated compounds were determined by off-line 1D and 2D NMR (COSY, HSQC, HMBC) and HPLC-ESI-MS. High-resolution MS was also applied for some isolated compounds. Absolute configurations were determined by ECD and NOESY. Optical rotation was recorded for chiral compounds.