Bacterial efflux inhibitors are widely distributed in land plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Secondary metabolites play a critical role in plant defense against disease and are of great importance to ethnomedicine. Bacterial efflux pumps are active transport proteins that bacterial cells use to protect themselves against multiple toxic compounds, including many antimicrobials. Efflux pump inhibitors from plants can block these efflux pumps, increasing the potency of antimicrobial compounds. This study demonstrates that efflux pump inhibition against the Gram-positive bacterial pathogen Staphylococcus aureus is widespread in extracts prepared from individual species throughout the land plant lineage. It therefore suggests a general mechanism by which plants used by indigenous species may be effective as a topical treatment for some bacterial infections. AIM OF THE STUDY: The goal of this research was to evaluate the distribution of efflux pump inhibitors in nine plant extracts with an ethnobotanical use suggestive of an antimicrobial function for the presence of efflux pump inhibitory activity against Staphylococcus aureus. MATERIALS AND METHODS: Plants were collected, dried, extracted, and vouchers submitted to the Herbarium of the University of North Carolina Chapel Hill (NCU). The extracts were analyzed by quantitative mass spectrometry (UPLC-MS) to determine the presence and concentration of flavonoids with known efflux pump inhibitory activity. A mass spectrometry-based assay was employed to measure efflux pump inhibition for all extracts against Staphylococcus aureus. The assay relies on UPLC-MS measurement of changes in ethidium concentration in the spent culture broth when extracts are incubated with bacteria. RESULTS: Eight of these nine plant extracts inhibited toxic compound efflux at concentrations below the MIC (minimum inhibitory concentration) value for the same extract. The most active extracts were those prepared from Osmunda claytoniana L. and Pinus strobes L., which both demonstrated IC50 values for efflux inhibition of 19 ppm. CONCLUSIONS: Our findings indicate that efflux pump inhibitors active against Staphylococcus aureus are common in land plants. By extension, this activity is likely to be important in many plant-derived antimicrobial extracts, including those used in traditional medicine, and evaluation of efflux pump inhibition may often be valuable when studying natural product efficacy.

Pharmacology in translation: the preclinical and early clinical profile of the novel α2/3 functionally selective GABAA receptor positive allosteric modulator PF-06372865.

BACKGROUND AND PURPOSE: Benzodiazepines, non-selective positive allosteric modulators (PAMs) of GABAA receptors, have significant side effects that limit their clinical utility. As many of these side effects are mediated by the α1 subunit, there has been a concerted effort to develop α2/3 subtype-selective PAMs. EXPERIMENTAL APPROACH: In vitro screening assays were used to identify molecules with functional selectivity for receptors containing α2/3 subunits over those containing α1 subunits. In vivo receptor occupancy (RO) was conducted, prior to confirmation of in vivo α2/3 and α1 pharmacology through quantitative EEG (qEEG) beta frequency and zolpidem drug discrimination in rats respectively. PF-06372865 was then progressed to Phase 1 clinical trials. KEY RESULTS: PF-06372865 exhibited functional selectivity for those receptors containing α2/3/5 subunits, with significant positive allosteric modulation (90-140%) but negligible activity (≤20%) at GABAA receptors containing α1 subunits. PF-06372865 exhibited concentration-dependent occupancy of GABAA receptors in preclinical species. There was an occupancy-dependent increase in qEEG beta frequency and no generalization to a GABAA α1 cue in the drug-discrimination assay, clearly demonstrating the lack of modulation at the GABAA receptors containing an α1 subtype. In a Phase 1 single ascending dose study in healthy volunteers, evaluation of the pharmacodynamics of PF-06372865 demonstrated a robust increase in saccadic peak velocity (a marker of α2/3 pharmacology), increases in beta frequency qEEG and a slight saturating increase in body sway. CONCLUSIONS AND IMPLICATIONS: PF-06372865 has a unique clinical pharmacology profile and a highly predictive translational data package from preclinical species to the clinical setting.

Secondary Metabolites from the Leaves of the Medicinal Plant Goldenseal (Hydrastis canadensis).

The study presented herein constitutes an extensive investigation of constituents in Hydrastis canadensis L. (Ranunculaceae) leaves. It describes the isolation and identification of two previously unknown compounds, 3,4-dimethoxy-2-(methoxycarbonyl)benzoic acid (1) and 3,5,3'-trihydroxy-7,4'-dimethoxy-6,8-C-dimethyl-flavone (2), along with the known compounds (±)-chilenine (3), (2R)-5,4'-dihydroxy-6-C-methyl-7-methoxy-flavanone (4), 5,4'-dihydroxy-6,8-di-C-methyl-7-methoxy-flavanone (5), noroxyhydrastinine (6), oxyhydrastinine (7) and 4',5'-dimethoxy-4-methyl-3'-oxo-(1,2,5,6-tetrahydro-4H-1,3-dioxolo-[4',5':4,5]-benzo[1,2-e]-1,2-oxazocin)-2-spiro-1'-phtalan (8). Compounds 3-8 have been reported from other sources, but this is the first report of their presence in H. canadensis extracts. A mass spectrometry based assay was employed to demonstrate bacterial efflux pump inhibitory activity against Staphylococcus aureus for 2, with an IC50 value of 180 ± 6 µM. This activity in addition to that of other bioactive compounds such as flavonoids and alkaloids, may explain the purported efficacy of H. canadensis for treatment of bacterial infections. Finally, this report includes high mass accuracy fragmentation spectra for all compounds investigated herein which were uploaded into the Global Natural Products Social molecular networking library and can be used to facilitate their future identification in H. canadensis or other botanicals.

A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.

Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 µg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 µg/mL to 75 µg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.

Developmentally regulated neurosteroid synthesis enhances GABAergic neurotransmission in mouse thalamocortical neurones.

KEY POINTS: During neuronal development synaptic events mediated by GABAA receptors are progressively reduced in their duration, allowing for rapid and precise network function. Here we focused on ventrobasal thalamocortical neurones, which contribute to behaviourally relevant oscillations between thalamus and cortex. We demonstrate that the developmental decrease in the duration of inhibitory phasic events results predominantly from a precisely timed loss of locally produced neurosteroids, which act as positive allosteric modulators of the GABAA receptor. The mature thalamus retains the ability to synthesise neurosteroids, thus preserving the capacity to enhance both phasic and tonic inhibition, mediated by synaptic and extrasynaptic GABAA receptors, respectively, in physiological and pathophysiological scenarios associated with perturbed neurosteroid levels. Our data establish a potent, endogenous mechanism to locally regulate the GABAA receptor function and thereby influence thalamocortical activity. During brain development the duration of miniature inhibitory postsynaptic currents (mIPSCs) mediated by GABAA receptors (GABAA Rs) progressively reduces, to accommodate the temporal demands required for precise network activity. Conventionally, this synaptic plasticity results from GABAA R subunit reorganisation. In particular, in certain developing neurones synaptic α2-GABAA Rs are replaced by α1-GABAA Rs. However, in thalamocortical neurones of the mouse ventrobasal (VB) thalamus, the major alteration to mIPSC kinetics occurs on postnatal (P) day 10, some days prior to the GABAA R isoform change. Here, whole-cell voltage-clamp recordings from VB neurones of mouse thalamic slices revealed that early in postnatal development (P7-P8), the mIPSC duration is prolonged by local neurosteroids acting in a paracrine or autocrine manner to enhance GABAA R function. However, by P10, this neurosteroid 'tone' rapidly dissipates, thereby producing brief mIPSCs. This plasticity results from a lack of steroid substrate as pre-treatment of mature thalamic slices (P20-24) with the GABAA R-inactive precursor 5α-dihydroprogesterone (5α-DHP) resulted in markedly prolonged mIPSCs and a greatly enhanced tonic conductance, mediated by synaptic and extrasynaptic GABAA Rs, respectively. In summary, endogenous neurosteroids profoundly influence GABAergic neurotransmission in developing VB neurones and govern a transition from slow to fast phasic synaptic events. Furthermore, the retained capacity for steroidogenesis in the mature thalamus raises the prospect that certain physiological or pathophysiological conditions may trigger neurosteroid neosynthesis, thereby providing a local mechanism for fine-tuning neuronal excitability.

Extrasynaptic GABA(A) receptors couple presynaptic activity to postsynaptic inhibition in the somatosensory thalamus.

Thalamocortical circuits govern cognitive, sensorimotor, and sleep-related network processes, and generate pathological activities during absence epilepsy. Inhibitory control of thalamocortical (TC) relay neurons is partially mediated by GABA released from neurons of the thalamic reticular nucleus (nRT), acting predominantly via synaptic α1ß2γ2 GABA(A) receptors (GABA(A)Rs). Importantly, TC neurons also express extrasynaptic α4ß2δ GABA(A)Rs, although how they cooperate with synaptic GABA(A)Rs to influence relay cell inhibition, particularly during physiologically relevant nRT output, is unknown. To address this question, we performed paired whole-cell recordings from synaptically coupled nRT and TC neurons of the ventrobasal (VB) complex in brain slices derived from wild-type and extrasynaptic GABA(A)R-lacking, α4 "knock-out" (α4(0/0)) mice. We demonstrate that the duration of VB phasic inhibition generated in response to nRT burst firing is greatly reduced in α4(0/0) pairs, suggesting that action potential-dependent phasic inhibition is prolonged by recruitment of extrasynaptic GABA(A)Rs. Furthermore, the influence of nRT tonic firing frequency on VB holding current is also greatly reduced in α4(0/0) pairs, implying that the α4-GABA(A)R-mediated tonic conductance of relay neurons is dynamically influenced, in an activity-dependent manner, by nRT tonic firing intensity. Collectively, our data reveal that extrasynaptic GABA(A)Rs of the somatosensory thalamus do not merely provide static tonic inhibition but can also be dynamically engaged to couple presynaptic activity to postsynaptic excitability. Moreover, these processes are highly sensitive to the δ-selective allosteric modulator, DS2 and manipulation of GABA transport systems, revealing novel opportunities for therapeutic intervention in thalamocortical network disorders.