Pharmacological Characterization of a Betaine/GABA Transporter 1 (BGT1) Inhibitor Displaying an Unusual Biphasic Inhibition Profile and Anti-seizure Effects.

Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 µM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.

Discovery of Potent Inhibitors for the Large Neutral Amino Acid Transporter 1 (LAT1) by Structure-Based Methods.

The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically diverse and potent inhibitors of LAT1. First, a homology model of LAT1 that is based on the atomic structures of the prokaryotic homologs was constructed. Molecular docking of nitrogen mustards (NMs) with a wide range of affinity allowed for deriving a common binding mode that could explain the structure-activity relationship pattern in NMs. Subsequently, validated binding hypotheses were subjected to molecular dynamics simulation, which allowed for extracting a set of dynamic pharmacophores. Finally, a library of ~1.1 million molecules was virtually screened against these pharmacophores, followed by docking. Biological testing of the 30 top-ranked hits revealed 13 actives, with the best compound showing an IC50 value in the sub-µM range.

Legacy data sharing to improve drug safety assessment: the eTOX project.

The sharing of legacy preclinical safety data among pharmaceutical companies and its integration with other information sources offers unprecedented opportunities to improve the early assessment of drug safety. Here, we discuss the experience of the eTOX project, which was established through the Innovative Medicines Initiative to explore this possibility.

Virtual Screening of DrugBank Reveals Two Drugs as New BCRP Inhibitors.

The breast cancer resistance protein (BCRP) is an ABC transporter playing a crucial role in the pharmacokinetics of drugs. The early identification of substrates and inhibitors of this efflux transporter can help to prevent or foresee drug-drug interactions. In this work, we built a ligand-based in silico classification model to predict the inhibitory potential of drugs toward BCRP. The model was applied as a virtual screening technique to identify potential inhibitors among the small-molecules subset of DrugBank. Ten compounds were selected and tested for their capacity to inhibit mitoxantrone efflux in BCRP-expressing PLB985 cells. Results identified cisapride (IC50 = 0.4 µM) and roflumilast (IC50 = 0.9 µM) as two new BCRP inhibitors. The in silico strategy proved useful to prefilter potential drug-drug interaction perpetrators among a database of small molecules and can reduce the amount of compounds to test.

A eudesmane-type sesquiterpene isolated from Pluchea odorata (L.) Cass. combats three hallmarks of cancer cells: Unrestricted proliferation, escape from apoptosis and early metastatic outgrowth in vitro.

Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC50 = 8.9 µM after 72 h) and 10 µM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100 µM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25 µM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.

The ABC of Phytohormone Translocation.

ATP-driven transport across biological membranes is a key process to translocate solutes from the interior of the cell to the extracellular environment. In humans, ATP-binding cassette transporters are involved in absorption, distribution, metabolism, excretion, and toxicity, and also play a major role in anticancer drug resistance. Analogous transporters are also known to be involved in phytohormone translocation. These include, e.g., the transport of auxin by ABCB1/19 in Arabidopsis thaliana, the transport of abscisic acid by AtABCG25, and the transport of strigolactone by the Petunia hybrida ABC transporter PDR1. Within this article, we outline the current knowledge about plant ABC transporters with respect to their structure and function, and provide, for the first time, a protein homology model of the strigolactone transporter PDR1 from P. hybrida.

Novel scaffolds for modulation of TRPV1 identified with pharmacophore modeling and virtual screening.

AIM: The transient receptor potential vanilloid type 1 (TRPV1) is responsible for pain perception in the peripheral nervous system (PNS). TRPV1 is thus considered a versatile target for development of non-opioid analgesics. RESULTS: Pharmacophore-based clustering of a publicly available data set of TRPV1 antagonists revealed a set of models, which were validated with data sets of inactive compounds, decoys and known drug candidates. The top ranked pharmacophore models were subsequently used for virtual screening. Based on a unique in-house protocol, a set of compounds was selected and biologically tested for modulation of TRPV1 in a voltage-clamp model. CONCLUSION: Pharmacophore models extracted from large public data sets are a valuable source for identification of novel scaffolds for TRPV1 receptor modulation.

The application of the open pharmacological concepts triple store (open PHACTS) to support drug discovery research.

Integration of open access, curated, high-quality information from multiple disciplines in the Life and Biomedical Sciences provides a holistic understanding of the domain. Additionally, the effective linking of diverse data sources can unearth hidden relationships and guide potential research strategies. However, given the lack of consistency between descriptors and identifiers used in different resources and the absence of a simple mechanism to link them, gathering and combining relevant, comprehensive information from diverse databases remains a challenge. The Open Pharmacological Concepts Triple Store (Open PHACTS) is an Innovative Medicines Initiative project that uses semantic web technology approaches to enable scientists to easily access and process data from multiple sources to solve real-world drug discovery problems. The project draws together sources of publicly-available pharmacological, physicochemical and biomolecular data, represents it in a stable infrastructure and provides well-defined information exploration and retrieval methods. Here, we highlight the utility of this platform in conjunction with workflow tools to solve pharmacological research questions that require interoperability between target, compound, and pathway data. Use cases presented herein cover 1) the comprehensive identification of chemical matter for a dopamine receptor drug discovery program 2) the identification of compounds active against all targets in the Epidermal growth factor receptor (ErbB) signaling pathway that have a relevance to disease and 3) the evaluation of established targets in the Vitamin D metabolism pathway to aid novel Vitamin D analogue design. The example workflows presented illustrate how the Open PHACTS Discovery Platform can be used to exploit existing knowledge and generate new hypotheses in the process of drug discovery.

In silico screening with benzofurane- and benzopyrane-type MDR-modulators.

Development of inhibitors of the drug efflux pump P-glycoprotein is a versatile approach to overcome multi drug resistance (MDR) in tumor therapy. In an approach to lower the conformational flexibility of the lead compound propafenone, we synthesized a set of dihydrobenzofuranes and benzopyranones. In the case of the 4 diastereomeric dihydrobenzofuranes, no significant differences in activity regarding the configuration on the side-chains at the dihydrofurane moiety (cis or trans) was observed. This may be due to the high flexibility of the side-chains, which still allow mutually overlap of pharmacophores. The benzopyranones showed a good correlation between lipophilicity and activity with gnerally lower logpotency/logP ratios. This decrease may be due to the rigidization of the molecules. In an in silico screening approach, a set of diverse propafenone-type compounds was used to establish a pharmacophore model, which was used to screen the world drug index. Among the hits retrieved there are several compounds, which were previously described as MDR-modulators. This demonstrates the validity of the model.