Mono-2-ethylhexylphthalate (MEHP) is a potent agonist of human TRPA1 channel.

Phthalates are extensively used as plasticizers in diverse consumer care products but have been reported to cause adverse health effects in humans. A commonly used phthalate, di-2-ethylhexylphthalate (DEHP) causes developmental and reproductive toxicities in humans, but the associated molecular mechanisms are not fully understood. Mono-2-ethylhexylphthalate (MEHP), a hydrolytic product of DEHP generated by cellular esterases, is proposed to be the active toxicant. We conducted a screen for sensory irritants among compounds used in consumer care using an assay for human Transient Receptor Potential A1 (hTRPA1). We have identified MEHP as a potent agonist of hTRPA1. MEHP-induced hTRPA1 activation was blocked by the TRPA1 inhibitor A-967079. Patch clamp assays revealed that MEHP induced inward currents in cells expressing hTRPA1. In addition, the N855S mutation in hTRPA1 associated with familial episodic pain syndrome decreased MEHP-induced hTRPA1 activation. In summary, we report that MEHP is a potent agonist of hTRPA1 which generates new possible mechanisms for toxic effects of phthalates in humans.

Inhibitors of human bitter taste receptors from the five-flavour berry, Schisandra chinensis.

The human bitter taste 2 receptor member 16 (TAS2R16) is one of 25 class A G-protein-coupled receptors (GPCRs) and responds to a variety of molecules responsible for the bitter taste sensation perceived in humans. TAS2R16 can be activated by ß-glucopyranosides, and its activation can be inhibited by probenecid, a synthetic drug compound used to treat gout. In this study we describe naturally derived compounds which can inhibit the activation of TAS2R16 by salicin in vitro. These compounds belong to the lignan class derived from the fruit of Schisandra chinensis, which is commonly known as the five-flavour berry. We further tested other analogs with this lignan scaffold, found their differential inhibitory activities towards TAS2R16 in vitro, and sought to rationalize the activity using molecular docking of these lignans on a computationally modelled structure of TAS2R16. Selected lignans with inhibitory activity against other TAS2Rs reveal sub-millimolar inhibitory activity towards TAS2R10, TAS2R14, and TAS2R43 in cell-based assays. These compounds with demonstrated in vitro inhibition of bitter taste receptors may serve as tool compounds to investigate the molecular mechanisms of hTAS2Rs biology in gustatory and non-gustatory tissues.

Construction of a bioluminescence-based assay for bitter taste receptors (TAS2Rs).

In humans, a family of 25 bitter taste receptors (TAS2Rs) mediates bitter taste perception. A common approach to characterize bitter causative agents involves expressing TAS2Rs and the appropriate signal transducers in heterologous cell systems, and monitoring changes in the intracellular free calcium levels upon ligand exposure using a fluorescence-based modality, which typically suffers from a low signal window, and is susceptible to interference by autofluorescence, therefore prohibiting its application to screening of plant or food extracts, which are likely to contain autofluorescent compounds. The aim of this study is to develop and validate a bioluminescence-based intracellular calcium release assay for TAS2Rs that has a better assay performance than a fluorescence-based assay. Furthermore, the bioluminescence-based assay enabled the evaluation of TAS2R agonists within an autofluorescent matrix, highlighting its potential utility in the assessment of the bitterness-inducing properties of plant or food fractions by the food industry. Additionally, improvement to the bioluminescence-based assay for some TAS2Rs was achieved by altering their N-terminal signal sequences, leading to signal window enhancement. Altogether, the bioluminescence-based TAS2R assay can be used to perform functional studies of TAS2Rs, evaluate TAS2R-modulating properties of autofluorescent samples, and facilitate the discovery of compounds that can function as promising bitter taste modulators.

2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 1. Identification of an Allosteric Binding Site.

CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non-ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD NMR, circular dichroism spectroscopy, and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC50 of 3.4 µM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anticancer drugs.

Evaluation of anti-proliferative activity of medicinal plants used in Asian Traditional Medicine to treat cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The extensive biodiversity of plants in Southeast Asia and inadequate research hitherto warrant a continued investigation into medicinal plants. On the basis of a careful review of fresh medicinal plant usage to treat cancer from previous ethnobotanical interviews in Singapore and from the traditional uses of the indigenous plants, fresh leaves of seven locally grown medicinal plant species were evaluated for anti-proliferative activity. AIM OF THE STUDY: To evaluate the anti-proliferative activity of local medicinal plant species Clausena lansium Skeels, Clinacanthus nutans (Burm. f.) Lindau, Leea indica (Burm. f.) Merr., Pereskia bleo (Kunth) DC., Strobilanthes crispus (L.) Blume, Vernonia amygdalina Delile and Vitex trifolia L. MATERIALS AND METHOD: Fresh, healthy and mature leaves of the seven medicinal plants were harvested from various locations in Singapore and Malaysia for Soxhlet, ultrasonication and maceration extractions in three different solvents (water, ethanol and methanol). Cell proliferation assay using water soluble tetrazolium salt (WST-1) assay was performed on twelve human cancer cell lines derived from breast (MDA-MB-231, T47D), cervical (C33A), colon (HCT116), leukemia (U937), liver (HepG2, SNU-182, SNU-449), ovarian (OVCAR-5, PA-1, SK-OV-3) and uterine (MES-SA/DX5) cancer. RESULTS: A total of 37 fresh leaf extracts from seven medicinal plants were evaluated for their anti-tumour activities in twelve human cancer cell lines. Of these, the extracts of C. lansium, L. indica, P. bleo, S. crispus, V. amygdalina and V. trifolia exhibited promising anti-proliferative activity against multiple cancer cell lines. Further investigation of selected promising leaf extracts indicated that maceration methanolic extract of L. indica was most effective overall against majority of the cancer cell lines, with best IC50 values of 31.5 ±â€¯11.4 µg/mL, 37.5 ±â€¯0.7 µg/mL and 43.0 ±â€¯6.2 µg/mL in cervical C33A, liver SNU-449, and ovarian PA-1 cancer cell lines, respectively. CONCLUSION: The results of this study provide new scientific evidence for the traditional use of local medicinal plant species C. lansium, L . indica, P. bleo, S. crispus, V. amygdalina and V. trifolia in cancer treatment. These results highlight the importance of the upkeep of these indigenous plants in modern society and their relevance as resources for drug discovery.

Structural insights into the EthR-DNA interaction using native mass spectrometry.

EthR is a transcriptional repressor that increases Mycobacterium tuberculosis resistance to ethionamide. In this study, the EthR-DNA interaction has been investigated by native electrospray-ionization mass spectrometry for the first time. The results show that up to six subunits of EthR are able to bind to its operator.

Disrupting the Constitutive, Homodimeric Protein-Protein Interface in CK2ß Using a Biophysical Fragment-Based Approach.

Identifying small molecules that induce the disruption of constitutive protein-protein interfaces is a challenging objective. Here, a targeted biophysical screening cascade was employed to specifically identify small molecules that could disrupt the constitutive, homodimeric protein-protein interface within CK2ß. This approach could potentially be applied to achieve subunit disassembly of other homo-oligomeric proteins as a means of modulating protein function.

Mass Spectrometry Reveals Protein Kinase CK2 High-Order Oligomerization via the Circular and Linear Assembly.

CK2 is an intrinsically active protein kinase that is crucial for cellular viability. However, conventional kinase regulatory mechanisms do not apply to CK2, and its mode of regulation remains elusive. Interestingly, CK2 is known to undergo reversible ionic-strength-dependent oligomerization. Furthermore, a regulatory mechanism based on autoinhibitory oligomerization has been postulated on the basis of the observation of circular trimeric oligomers and linear CK2 assemblies in various crystal structures. Here, we employ native mass spectrometry to monitor the assembly of oligomeric CK2 species in an ionic-strength-dependent manner. A subsequent combination of ion mobility spectrometry-mass spectrometry and hydrogen-deuterium exchange mass spectrometry techniques was used to analyze the conformation of CK2 oligomers. Our findings support ionic-strength-dependent CK2 oligomerization, demonstrate the transient nature of the α/ß interaction, and show that CK2 oligomerization proceeds via both the circular and linear assembly.

Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity.

Selected cytotoxic chemicals can provoke the immune system to recognize and destroy malignant tumors. Most of the studies on immunogenic cell death are focused on the signals that operate on a series of receptors expressed by dendritic cells to induce tumor antigen-specific T-cell responses. Here, we explored the effects of oxaliplatin, an immunogenic cell death inducer, on the induction of stress ligands and promotion of natural killer (NK) cell-mediated cytotoxicity in human ovarian cancer cells. The results indicated that treatment of tumor cells with oxaliplatin induced the production of type I interferons and chemokines and enhanced the expression of major histocompatibility complex class I-related chains (MIC) A/B, UL16-binding protein (ULBP)-3, CD155 and TNF-related apoptosis-inducing ligand (TRAIL)-R1/R2. Furthermore, oxaliplatin but not cisplatin treatment enhanced susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. In addition, activated NK cells completely abrogated the growth of cancer cells that were pretreated with oxaliplatin. However, cancer cells pretreated with the same concentration of oxaliplatin alone were capable of potentiating regrowth over a period of time. These results suggest an advantage in combining oxaliplatin and NK cell-based therapy in the treatment of ovarian cancer. Further investigation on such potential combination therapy is warranted.

Ethnobotanical survey of usage of fresh medicinal plants in Singapore.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of medicinal plants in human health has been documented since ancient times and they provide a useful source of new therapeutics. In Singapore, despite the accessibility to modern healthcare, there still exist pockets of the population who choose to use locally grown fresh medicinal plants for health promotion and even therapeutic purposes. However to date, there is no published report of first-hand account of their usage in Singapore. As land is scarce and rapidly used for re-development, such important knowledge may be lost if not properly documented in time. This work safeguards the local folk knowledge, and provides information on common and scarcely reported fresh medicinal plants. The objective of this study is to gather information regarding the usage of fresh medicinal plants in Singapore through face-to-face interviews. MATERIALS AND METHODS: Information on demographic data and plant-use methods were collated via face-to-face interviews of 200 fresh medicinal plant users who have used fresh medicinal plants in the last five years. The survey protocol was approved by the National University of Singapore Institutional Review Board and informed consent was obtained from every participant. RESULTS: A total of 414 plants represented by 104 plant species from 44 families were reportedly used by the 200 participants. The five most commonly used plants were Clinacanthus nutans (34 users), Strobilanthes crispus (31 users), Pereskia bleo (25 users), Aloe vera (18 users) and Zingiber officinale (16 users). Leaves were the most commonly used plant part while preparing a decoction was the most common method of preparation. The majority of interviewees used plants for general health purposes and to treat diseases related to the respiratory system and cancer. CONCLUSIONS: Our survey has successfully documented the rich wealth of traditional usage and knowledge on 414 fresh medicinal plants grown in Singapore through face-to-face interviews with 200 users. This study will serve as a useful resource for identifying promising plants for future drug discovery efforts. Scientific evaluations of these medicinal plants are warranted and should be carried out.

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques.

PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.