HPLC-based activity profiling for pharmacologically and toxicologically relevant natural products - principles and recent examples.

CONTEXT: Discovery of pharmacologically active natural products as starting points for drug development remains important and, for reasons of consumer safety, the identification of toxicologically relevant compounds in herbal drugs. OBJECTIVE: To explain, with the aid of relevant examples from our own research, how these goals can be achieved. METHODS: An in-house technology platform comprising pre-formatted extract libraries in 96-well format, miniaturized tracking of activity in extracts via HPLC-activity profiling, structure elucidation with microprobe NMR, and in vitro and in vivo pharmacological methods were used. RESULTS: Piperine was identified as a new scaffold for allosteric GABAA receptor modulators with in vivo activity that interacts at a benzodiazepine-independent binding site. Selectivity and potency were improved by iterative optimization towards synthetic piperine analogues. Dehydroevodiamine and hortiamine from the traditional Chinese herbal drug Evodiae fructus were identified as potent hERG channel blockers in vitro. The compounds induced torsades de pointes arrhythmia in animal models. CONCLUSIONS: The allosteric binding site for piperine analogues remains to be characterized and cardiac risks of herbal drugs need to be further evaluated to ensure consumer safety.

Disruption of brain MEK-ERK sequential phosphorylation and activation during midazolam-induced hypnosis in mice: Roles of GABAA receptor, MEK1 inactivation, and phosphatase MKP-3.

Midazolam is a positive allosteric modulator at GABAA receptor that induces a short hypnosis and neuroplasticity, in which the sequential phosphorylation of MEK1/2 and ERK1/2 was shown to play a role. This study investigated the parallel activation of p-MEK and p-ERK and regulatory mechanisms induced by midazolam through the stimulation of GABAA receptors in the mouse brain. During the time course of midazolam (60mg/kg)-induced sleep in mice (lasting for about 2h) p-Ser217/221 MEK1/2 was increased (+146% to +258%) whereas, unexpectedly, p-Tyr204/Thr202 ERK1/2 was found decreased (-16% to -38%), revealing uncoupling of MEK to ERK signals in various brain regions. Midazolam-induced p-MEK1/2 upregulation was prevented by pretreatment (30min) with flumazenil (10mg/kg), indicating the involvement of GABAA receptors. Also unexpectedly, midazolam-induced p-ERK1/2 downregulation was not prevented by flumazenil (10 or 30mg/kg). Notably, during midazolam-induced sleep the content of inactivated p-Thr286 MEK1, which can dampen ERK1/2 activation, was increased (+33% to +149%) through a mechanism sensitive to flumazenil (10mg/kg). Midazolam also increased MKP-3 (+13% to +73%) content and this upregulation was prevented by flumazenil (10mg/kg); an effect suggesting ERK inactivation because MKP-3 is the phosphatase selective for ERK1/2 dephosphorylation. The results indicate that during midazolam-induced sleep in mice there is an uncoupling of p-MEK (increased) to p-ERK (decreased) signals. p-ERK1/2 downregulation (not involving GABAA receptors) is the result of increased inactivated MEK1 and phosphatase MKP-3 (both effects involving GABAA receptors). These findings are relevant for the neurobiology and clinical use of benzodiazepines.

Design, Synthesis and Evaluation of the Antidepressant and Anticonvulsant Activities of Triazole-Containing Benzo[d]oxazoles.

BACKGROUND: Epilepsy and depression are two of the common diseases seriously threatening life and health of human. A shared neurobiological substrate led to the bidirectional relationship and high comorbid occurrence of the two disorders. Recently, an increasing number of patients with epilepsy (PWE) require some form of antidepressant medication. However, most of the available antidepressants are inadequate for PWE for some reasons. So, the search for novel and increasingly effective drugs with anticonvulsant and antidepressant activities is necessary. METHODS: A series of 2-substituted-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazoles (5a-p) were designed and synthesized. Their anticonvulsant activities were evaluated using maximal electroshock shock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice. Their antidepressant activities were screened with the forced swimming test (FST). RESULTS: All the compounds showed anti-MES activities in different degree, among which 5g and 5j were the most promising one with ED50 value of 31.7 and 12.7 mg/kg, respectively. What's more, 5g and 5j also exhibited nice anti-scPTZ activities and low neurotoxicity. Interestingly, these compounds also showed good antidepressant activities in FST. And the efficacy of 5g were also confirmed by a tail suspension test and a open field test. The pretreatment of thiosemicarbazide (an inhibitor of γ- aminobutyric acid synthesis enzyme) significantly increased the ED50 of 5g in MES and reversed the reductions in the immobility time of 5g in FST. CONCLUSION: Triazole-containing benzo[d]oxazole is a good skeleton to develop compounds with both anticonvulsant and antidepressant activities. We have got the compound 5g, which display remarkable antidepressant and anticonvulsant activities, and the GABAergic system was involved in the action mechanism of 5g.

Reversal of prenatal diazepam-induced deficit in a spatial-object learning task by brief, periodic maternal separation in adult rats.

In the rat, prenatal exposure to diazepam (DZ) induces a permanent reduction in GABA/BZ receptor (R) function and behavioural abnormalities. Environmental modifications during early stages of life can influence brain development and induce neurobiological and behavioural changes throughout adulthood. Indeed, a subtle, periodic, postnatal manipulation increases GABA/BZ R activity and produces facilitatory effects on neuroendocrine and behavioural responses. We here investigated the impact of prenatal treatment with DZ on learning performance in adult 3- and 8-month-old male rats and the influence of a brief, periodic maternal separation on the effects exerted by prenatal DZ exposure. Learning performance was examined employing a non-aversive spatial, visual and/or tactile task, the "Can test". Behavioural reactivity, emotional state and fear/anxiety-driven behaviour were also examined using open field (OF), acoustic startle reflex (ASR) and elevated plus-maze (EPM) tests. A single daily injection of DZ (1.5mg/kg, s.c.), over gestational days (GD) 14-20, induced, in an age-independent manner, a severe deficit in learning performance, a decrease in locomotor and explorative activity and an increase in peak amplitude in the ASR. Furthermore, anxiety-driven behaviour in EPM was disrupted. Daily maternal separation for 15 min over postnatal days 2-21 exerted opposite effects in all the paradigms examined. Prenatally DZ-exposed maternal separated rats, in contrast to respective non-separated rats, showed an improvement in learning performance, a decrease in emotionality and a normalization of the exploratory behaviour in EPM. These results suggest that a greater maternal care, induced by separation, can serve as a source for the developing brain to enhance neuronal plasticity and to prevent the behavioural abnormalities induced by prenatal DZ exposure.