Esther Omaiye, Kevin J McWhirter, Wentai Luo, James F Pankow, Prue Talbot

While JUUL electronic cigarettes (ECs) have captured the majority of the EC market with a large fraction of their sales going to adolescents, little is known about their cytotoxicity and potential effects on health. The purpose of this study was to determine flavor chemical and nicotine concentrations in the eight currently marketed pre-filled JUUL EC cartridges (pods) and to evaluate the cytotoxicity of the different variants (e.g., Cool Mint and Creme Brulee) using in vitro assays. Nicotine and flavor chemicals were analyzed using gas chromatography/mass spectrometry in pod fluid before and after vaping and in the corresponding aerosols. 59 flavor chemicals were identified in JUUL pod fluids, and three were >1 mg/mL. Duplicate pods were similar in flavor chemical composition and concentration. Nicotine concentrations (average 60.9 mg/mL) were significantly higher than any EC products we have analyzed previously. Transfer efficiency of individual flavor chemicals that were >1mg/mL and nicotine from the pod fluid into aerosols was generally 35 - 80%. All pod fluids were cytotoxic at a 1:10 dilution (10%) in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. Most aerosols were cytotoxic in these assays at concentrations >1%. The cytotoxicity of aerosols was highly correlated with nicotine and ethyl maltol concentrations and moderately to weakly correlated with total flavor chemical concentration and menthol concentration. Our study demonstrates that: (1) some JUUL flavor pods have high concentrations of flavor chemicals that may make them attractive to youth, and (2) the concentrations of nicotine and some flavor chemicals (e.g. ethyl maltol) are high enough to be cytotoxic in acute in vitro assays, emphasizing the need to determine if JUUL products will lead to adverse health effects with chronic use.

Regina R. Monaco, Rena F. Quinlan

Abstract: Discovery of novel natural products is an accepted method for the elucidation of pharmacologically active molecules and drug leads. Best known sources for such discovery have been terrestrial plants and microbes, accounting for about 85% of the approved natural products in pharmaceutical use (1), and about 60% of approved pharmaceuticals and new drug applications annually (2). Discovery in the marine environment has lagged due to the difficulty of exploration in this ecological niche. Exploration began in earnest in the 1950’s, after technological advances such as scuba diving allowed collection of marine organisms, primarily at a depth to about 15m. Natural products from filter feeding marine invertebrates and in particular, sponges, have proven to be a rich source of structurally unique pharmacologically active compounds, with over 16,000 molecules isolated thus far (3, 1) and a continuing pace of discovery at hundreds of novel bioactive molecules per year. All classes of pharmaceuticals have been represented in this discovery process, including antiprotazoals, pesticides, TGF-beta inhibitors, cationic channel blockers, anticancer, cytotoxic, antiviral, anti-inflammatory and antibacterial compounds. Important biosynthetic pathways found in sponges which give rise to these compounds include the terpenoid (4), fatty acid, polyketoid, quinone reductase, alkaloid, isoprenoid (5), and non-ribosomal protein synthase pathways. Keywords: natural products; marine sponges; drug discovery; terpenoids; carotenoids; polyketides; marine drug discovery

Qiuxia Fan, Xiaoming Feng, Xizhen Hong, Siqiao Gong, Jianwei Tian, Fanfan Hou, Fujian Zhang

Kidney stone formers with family history have a high rate of stone recurrence after kidney stone removal surgery and there is no effective medication available for treatment. Here, we show that Garcinia cambogia extract (GCE) efficiently removes calcium oxalate kidney stones from Malpighian tubules in both genetic and non-genetic Drosophila models of nephrolithiasis, and hydroxycitrate -a major component of GCE, directly dissolves calcium oxalate stones in Drosophila Malpighian tubules ex vivo. Our study discovers a potential novel therapeutic strategy for the clinical treatment of nephrolithiasis and suggests that clinical-grade Garcinia cambogia extract could be used to treat patients with nephrolithiasis in the future.

Neel S. Madhukar, Prashant K. Khade, Linda Huang, Kaitlyn Gayvert, Giuseppe Galletti, Martin Stogniew, Joshua E. Allen, Paraskevi Giannakakou, Olivier Elemento

Drug target identification is one of the most important aspects of pre-clinical development yet it is also among the most complex, labor-intensive, and costly. This represents a major issue, as lack of proper target identification can be detrimental in determining the clinical application of a bioactive small molecule. To improve target identification, we developed BANDIT, a novel paradigm that integrates multiple data types within a Bayesian machine-learning framework to predict the targets and mechanisms for small molecules with unprecedented accuracy and versatility. Using only public data BANDIT achieved an accuracy of approximately 90% over 2000 different small molecules - substantially better than any other published target identification platform. We applied BANDIT to a library of small molecules with no known targets and generated ~4,000 novel molecule-target predictions. From this set we identified and experimentally validated a set of novel microtubule inhibitors, including three with activity on cancer cells resistant to clinically used anti-microtubule therapies. We next applied BANDIT to ONC201 - an active anti-cancer small molecule in clinical development - whose target has remained elusive since its discovery in 2009. BANDIT identified dopamine receptor 2 as the unexpected target of ONC201, a prediction that we experimentally validated. Not only does this open the door for clinical trials focused on target-based selection of patient populations, but it also represents a novel way to target GPCRs in cancer. Additionally, BANDIT identified previously undocumented connections between approved drugs with disparate indications, shedding light onto previously unexplained clinical observations and suggesting new uses of marketed drugs. Overall, BANDIT represents an efficient and highly accurate platform that can be used as a resource to accelerate drug discovery and direct the clinical application of small molecule therapeutics with improved precision.

Manuel Rueda, Ruben Abagyan

During the last decade we witnessed how computational docking methods became a crucial tool in the search for new drug candidates. The 'central dogma' of small molecule docking is that compounds that dock correctly into the receptor are more likely to display biological activity than those that do not dock. This 'dogma', however, possesses multiple twists and turns that may not be obvious to novice dockers. The first premise is that the compounds must dock; this implies: (i) availability of data, (ii) realistic representation of the chemical entities in a form that can be understood by the computer and the software, and, (iii) exhaustive sampling of the protein-ligand conformational space. The second premise is that, after the sampling, all docking solutions must be ranked correctly with a score representing the physico-chemical foundations of binding. The third premise is that 'correctness' must be defined unambiguously, usually by comparison with 'static' experimental data (or lack thereof). Each of these premises involves some degree of simplification of reality, and overall loss in the accuracy of the docking predictions. In this chapter we will revise our latest experiences in receptor-based docking when dealing with all three above-mentioned issues. First, we will explain the theoretical foundation of ICM docking, along with a brief explanation on how we measure performance. Second, we will contextualize ICM by showing its performance in single and multiple receptor conformation schemes with the Directory of Useful Decoys (DUD) and the Pocketome. Third, we will describe which strategies we are using to represent protein plasticity, like using multiple crystallographic structures or Monte Carlo (MC) and Normal Mode Analysis (NMA) sampling methods, emphasizing how to overcome the associated pitfalls (e.g., increased number of false positives). In the last section, we will describe ALiBERO, a new tool that is helping us to improve the discriminative power of X-ray structures and homology models in screening campaigns.

Jaak Simm, Günter Klambauer, Adam Arany, Marvin Steijaert, Jörg Kurt Wegner, Emmanuel Gustin, Vladimir Chupakhin, Yolanda T. Chong, Jorge Vialard, Peter Buijnsters, Ingrid Velter, Alexander Vapirev, Shantanu Singh, Anne Carpenter, Roel Wuyts, Sepp Hochreiter, Yves Moreau, Hugo Ceulemans

We repurpose a High-Throughput (cell) Imaging (HTI) screen of a glucocorticoid receptor assay to predict target protein activity in multiple other seemingly unrelated assays. In two ongoing drug discovery projects, our repurposing approach increased hit rates by 60- to 250-fold over that of the primary project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from available HTI screens are a rich source of information that can be reused to empower drug discovery efforts.

Thomas James Wood, Dave Goulson

EXECUTIVE SUMMARY Neonicotinoid pesticides were first introduced in the mid-1990s and since then their use has grown rapidly so that they have become the most widely used class of insecticides in the world, with the majority being used as seed coatings. Neonicotinoids are water-soluble, and so a small quantity applied to a seed will dissolve when in contact with water in the soil and be taken up by the roots of the developing plant. Once inside the plant it becomes systemic and is found in vascular tissues and foliage, providing protection against herbivorous insects. This prophylactic use of neonicotinoids has become extremely widespread on a wide range of arable crops across much of the developed world. However, only approximately 5% of the neonicotinoid active ingredient is taken up by crop plants and most instead disperses into the wider environment. Since the mid-2000s numerous studies have raised concerns that neonicotinoids may be having a negative effect on non-target organisms. In particular, neonicotinoids were associated with mass poisoning events of honeybees and were shown to have serious negative effects on honeybee and bumblebee fitness when consumed. In response to this growing body of evidence, the European Food Safety Authority (EFSA) was commissioned to produce risk assessments for the use of clothianidin, imidacloprid and thiamethoxam and their impact on bees. These risk assessments, published in January 2013, conclude that the use of these compounds on certain flowering crops poses a high risk to bees. On the basis of these findings, the European Union adopted a partial ban on these substances in May 2013 which came into force on 1st December 2013. The purpose of this review is to collate and summarise scientific evidence published since 2013 that investigates the impact of neonicotinoids on non-target organisms and to bring it into one place to aid informed decision making. Due to international concern over the unintended impacts of neonicotinoids on wildlife, this topic has received a great deal of scientific attention in this three year period. As the restrictions were put in place because of the risk neonicotinoids pose to bees, much of the recent research work has naturally focussed on this group. Risks to bees Broadly, the EFSA risk assessments addressed risks of exposure to bees from neonicotinoids through various routes and the direct lethal and sublethal impact of neonicotinoid exposure. New scientific evidence is available in all of these areas, and it is possible to comment on the change in the scientific evidence since 2013 compared to the EFSA reports. This process is not meant to be a formal assessment of the risk posed by neonicotinoids in the manner of that conducted by EFSA. Instead it aims to summarise how the new evidence has changed our understanding of the likely risks to bees; is it lower, similar or greater than the risk perceived in 2013. With reference to the EFSA 2013 risk assessments baseline, advances in each considered area and their impact on the original assessment can be summarised thus: * Risk of exposure from pollen and nectar of treated flowering crops. The EFSA reports calculated typical exposure from flowering crops treated with neonicotinoids as seed dressings. Considerably more data are now available in this area, with new studies broadly supporting the calculated exposure values. For bees, flowering crops pose a Risk Unchanged to that reported by EFSA 2013. * Risk from non-flowering crops and cropping stages prior to flowering. Non-flowering crops were considered to pose no risk to bees. No new studies have demonstrated that these non-flowering crops pose a direct risk to bees. They remain a Risk Unchanged. * Risk of exposure from the drilling of treated seed and subsequent dust drift. Despite modification in seed drilling technology, available studies suggest that dust drift continues to occur, and that dust drift still represents a source of acute exposure and so is best considered a Risk Unchanged. * Risk of exposure from guttation fluid. Based on available evidence this was considered a low-risk exposure path by EFSA 2013. New data have not changed this position and so it remains a Risk Unchanged. * Risk of exposure from and uptake of neonicotinoids in non-crop plants. Uptake of neonicotinoids by non-target plants was considered likely to be negligible, though a data gap was identified. Many studies have since been published demonstrating extensive uptake of neonicotinoids and their presence in the pollen, nectar and foliage of wild plants. Bees collecting pollen from neonicotinoid-treated crops can generally be expected to be exposed to the highest neonicotinoid concentrations, but non-trivial quantities of neonicotinoids are also present in pollen and nectar collected from wild plants, and this source of exposure may be much more prolonged than the flowering period of the crop. Exposure from non-target plants clearly represents a Greater Risk. * Risk of exposure from succeeding crops. A data gap was identified for this issue. Few studies have explicitly investigated this, but this area does represent some level of risk as neonicotinoids are now known to have the potential to persist for years in soil, and can be detected in crops multiple years after the last known application. However, as few data exist this is currently considered a Risk Unchanged. * Direct lethality of neonicotinoids to adult bees. Additional studies on toxicity to honeybees have supported the values calculated by EFSA. More data have been produced on neonicotinoid toxicity for wild bee species and meta-analyses suggest a broadly similar response. Reference to individual species is important but neonicotinoid lethality should be broadly considered a Risk Unchanged. * Sublethal effects of neonicotinoids on wild bees. Consideration of sublethal effects by EFSA was limited as there is no agreed testing methodology for the assessment of such effects. A data gap was identified. Exposure to neonicotinoid-treated flowering crops has been shown to have significant negative effects on free flying wild bees under field conditions and some laboratory studies continue to demonstrate negative effects on bee foraging ability and fitness using field-realistic neonicotinoid concentrations. Greater Risk. Within this context, research produced since 2013 suggest that neonicotinoids pose a similar to greater risk to wild and managed bees, compared to the state of play in 2013. Given that the initial 2013 risk assessment was sufficient to impose a partial ban on the use of neonicotinoids on flowering crops, and given that new evidence either confirms or enhances evidence of risk to bees, it is logical to conclude that the current scientific evidence supports the extension of the moratorium, and that the extension of the partial ban to other uses of neonicotinoids should be considered. Broader risks to environmental health In addition to work on bees, our scientific understanding has also been improved in the following areas which were not previously considered by EFSA: * Non-flowering crops treated with neonicotinoids can pose a risk to non-target organisms through increasing mortality in beneficial predator populations. * Neonicotinoids can persist in agricultural soils for several years, leading to chronic contamination and, in some instances, accumulation over time. * Neonicotinoids continue to be found in a wide range of different waterways including ditches, puddles, ponds, mountain streams, rivers, temporary wetlands, snowmelt, groundwater and in outflow from water processing plants. * Reviews of the sensitivity of aquatic organisms to neonicotinoids show that many aquatic insect species are several orders of magnitude more sensitive to these compounds than the traditional model organisms used in regulatory assessments for pesticide use. * Neonicotinoids have been shown to be present in the pollen, nectar and foliage of non-crop plants adjacent to agricultural fields. This ranges from herbaceous annual weeds to perennial woody vegetation. We would thus expect non-target herbivorous insects and non-bee pollinators inhabiting field margins and hedgerows to be exposed to neonicotinoids. Of particular concern, this includes some plants sown adjacent to agricultural fields specifically for the purposes of pollinator conservation. * Correlational studies have suggested a negative link between neonicotinoid usage in agricultural areas and population metrics for butterflies, bees and insectivorous birds in three different countries. Overall, this recent work on neonicotinoids continues to improve our understanding of how these compounds move through and persist in the wider environment. These water soluble compounds are not restricted to agricultural crops, instead permeating most parts of the agricultural environments in which they are used and in some cases reaching further afield via waterways and runoff water. Field-realistic laboratory experiments and field trials continue to demonstrate that traces of residual neonicotinoids can have a mixture of lethal and sublethal effects on a wide range of taxa. Susceptibility varies tremendously between different taxa across many orders of magnitude, with some showing a negative response at parts per billion with others show no such effects at many thousands of parts per billion. Relative to the risk assessments produced in 2013 for clothianidin, imidacloprid and thiamethoxam which focussed on their effects on bees, new research strengthens arguments for the imposition of a moratorium, in particular because it has become evident that they pose significant risks to many non-target organisms, not just bees. Given the improvement in scientific knowledge of how neonicotinoids move into the wider environment from all crop types, a discussion of the risks posed by their use on non-flowering crops and in non-agricultural areas is urgently needed.

Jeremy Horst, Jong Seto

The current paradigm of treatment for dental caries (tooth decay) in primary teeth is dangerous, fails to reach many children, and suffers high recurrence. Acceptance of the paradigm arises from a misperception that untreated caries in primary teeth is a threat to life. We show a linear relationship between age and deaths in the United States from 1999 through 2015 caused by dental caries, pulpal/periapical abscess, or facial cellulitis. The intercept of 6 years coincides with emergence of the first permanent tooth: it appears that caries in primary teeth is not a threat to life. Thus, treatment goals should be to avoid pain, which is not possible with operative dentistry, as it causes pain. Medical management of caries is a distinct treatment philosophy which employs topical minimally invasive therapies that treat the disease, and is not merely prevention. Silver diamine fluoride (SDF) is a central agent to enable effective non-invasive treatment. The announcement of FDA Breakthrough Therapy designation suggests that SDF will become the first FDA approved drug for treating the disease dental caries. Since our last review, 4 clinical trials have been completed, which inform an update to the application protocol and frequency regimen. Suggestions from these studies are to skip the rinsing step due to demonstration of safety and concern of diminished effectiveness by dilution, and to start patients with an intensive regimen of multiple applications over the first few weeks. Breakthroughs in elucidating the impact of SDF on tooth structure and the plaque microbiome inform potential opportunities for bioengineering and understanding caries arrest, respectively. Dentists have been surprised by preference of this treatment over traditional invasive approaches. Renewed interest in this old material has delivered progress to optimize the judicious use of SDF, and enable a revolution in caries management, particularly for primary teeth.

S. Feranchuk, U. Potapova, S. Belikov

The NS3 protease and NS5 polymerase structures of the Zika virus were constructed and processed using a virtual screening pipeline. MM-PBSA calculations show that some of the ligands found by the pipeline demonstrate a good affinity to vulnerable sites in both proteins. The anti-hypertension drug eprosartan is among the selected ligands; and inhibition of the tick-borne encephalitis virus has already been confirmed in vivo by a previous study. In the present study phytochemicals bisabolol and andrographolide are suggested as the candidates for antiviral therapy.

Maria Angelica M Duque, Rhowell N Tiozon, Rebecca C. Nueva España

Nanotechnology and its promises for clinical translation to targeted drug delivery with limited accompanying toxicity provide exciting research opportunities that demands multidisciplinary approaches. The colloidal metallic systems have been recently investigated in the area of nanomedicine. Gold nanoparticles have found themselves useful for diagnostics and drug delivery applications. In this study, we have reported a novel method for the synthesis of gold nanoparticles using natural, biocompatible and biodegradable chitosan which came from deacetylating chitin from Portunus Pelagicus. It serves many purposes, as a reducing agent, stabilizer and absorption and penetration enhancer. Erythropoietin would have high loading efficiency with chitosan reduced gold nanoparticles; the binding is predominantly through hydrogen bonding. Chitosan reduced gold nanoparticles improve the pharmacodynamics and cellular uptake of Erythropoietin across mucosal sites and have immunoadjuvant properties. There is almost 50 % shell waste in crustacean industry. It is resourceful if it would be bioconverted. The process of bioconversion is deproteination, demineralization and deacetylation to obtain chitosan. In synthesizing gold nanoparticles, 1.48 x 10-2 M chloroauric acid will be reduced by heating for 15 minutes in 100mL chitosan solution prepared in 1% acetic acid to yield a ruby-red solution. Erythropoietin would be loaded into it and will undergo 13,000rpm of centrifuge followed by calculating the loading efficiency.

Thomas Pokorny, Patricia Duerler, Erich Seifritz, Franz X. Vollenweider, Katrin H. Preller

Psychiatric and neurodegenerative illnesses are characterized by cognitive impairments, in particular deficits in working memory, decision making, and executive functions including cognitive flexibility. However, the neuropharmacology of these cognitive functions is poorly understood. The serotonin (5-HT) 2A receptor might be a promising candidate for the modulation of cognitive processes. However, pharmacological studies investigating the role of this receptor system in humans are rare. Recent evidence demonstrates that the effects of Lysergic acid diethylamide (LSD) are mediated via agonistic action at the 5-HT2A receptor. Yet, the effects of LSD on specific cognitive domains using standardized neuropsychological test have not been studied. Therefore, we examined the acute effects of LSD (100 micrograms) alone and in combination with the 5-HT2A antagonist ketanserin (40mg) on cognition, employing a double-blind, randomized, placebo-controlled, within-subject design in 25 healthy participants. Executive functions, cognitive flexibility, spatial working memory, and risk-based decision-making were examined by the Intra/Extra-Dimensional shift task (IED), Spatial Working Memory task (SWM), and Cambridge Gambling Task (CGT) of the Cambridge Neuropsychological Test Automated Battery. Compared to placebo, LSD significantly impaired executive functions, cognitive flexibility, and working memory on the IED and SWM, but did not influence quality of decision-making and risk-taking on the CGT. Pretreatment with the 5-HT2A antagonist ketanserin normalized all LSD-induced cognitive deficits. The present findings highlight the role of the 5-HT2A receptor system in executive functions and working memory and suggest that specific 5-HT2A antagonists may be relevant for improving cognitive dysfunctions in psychiatric disorders.

Rafael M Couñago, Alison D Axtman, Stephen J Capuzzi, Hátylas Azevedo, David H Drewry, Jonathan M. Elkins, Opher Gileadi, Cristiano R. W. Guimarães, Alessandra Mascarello, Ricardo A. M. Serafim, Carrow I Wells, Timothy M. Willson, William J Zuercher

Understanding the structural determinants of inhibitor selectivity would facilitate the design and preparation of kinase probes. We describe a pair of matched compounds differing only by one degree of saturation but showing dramatic differential activities at select kinases. We utilized x-ray crystallography and computational analysis to rationalize the basis of the differential activity.

Matthew J Baggott, Kathleen J Garrison

Hyponatremia is a serious complication of 3,4-methylenedioxymethamphetamine (MDMA) use. We investigated potential mechanisms in two double-blind, placebo-controlled studies. In study 1, healthy drug-experienced volunteers received MDMA or placebo alone and in combination with the alpha-1 adrenergic inverse agonist prazosin, used as a positive control to release antidiuretic hormone (ADH). In study 2, volunteers received MDMA or placebo followed by standardized water intake. MDMA lowered serum sodium, but did not increase ADH or copeptin, although the control prazosin did increase ADH. Water loading reduced serum sodium more after MDMA than after placebo. There was a trend for women to have lower baseline serum sodium than men, but there were no significant interactions with drug condition. Combining studies, MDMA potentiated the ability of water to lower serum sodium. Thus, hyponatremia appears to be a significant risk when hypotonic fluids are consumed during MDMA use. Clinical trials and events where MDMA use is common should anticipate and mitigate this risk.

Yuzheng Feng, Aryan Luthra, Kaiwen Ding, Yang Yang, Jordan Savage, Xinrui Wei, Roland Moeschter, Sachin Ahuja, Victor Villegas, Bogdana Torbina, Anjuli Ahooja, Tom Ellis

This study investigated the negative effects of neonicotinoid pesticides on honey bees in environment surrounding areas of pesticide use. The aim of the experiment is to identify possible contributors to the sudden decrease in honey bee population over the past 60 years, a phenomenon known as Colony Collapse Disorder. Analysis was performed on three sets of bees: the control group which was not in contact with pesticides, the infected dead group which was a set of bees suspected to have died due to neonicotinoids, and the infected alive group which was suspected to be under the influence of neonicotinoids. After dissecting the bee samples and extracting their fat bodies, the chemical composition and protein structures of the samples were analyzed using Mid-Infrared Beamline at the Canadian Light Source. Results from the spectra of bee samples exposed to neonicotinoids demonstrated possible residual pesticide chemicals within fat bodies. Several spectral peaks were also correlated with a possible change in protein secondary structures from primarily β-sheet to α-helix within fat bodies of neonicotinoid-affected bees. It is likely that the pesticides caused the growth of additional α-helical structures, which is consistent with consequences of the inhibition of nicotinic acetylcholine receptors (nAChRs), which is a pathway of harm of Colony Collapse Disorder as identified in past literature.

Alvaro Cortes Cabrera, Daniel Lucena-Agell, Mariano Redondo-Horcajo, Isabel Barasoain, Fernando Diaz, Bernhard Fasching, Paula Petrone

We developed a statistical method, BIOSEA, able to identify molecules that are capable of reproducing a desired cellular phenotype, by scanning a large compound collection based on biological similarity. Our method leverages highly incomplete and noisy compound bioactivity signatures from historical high-throughput screening campaigns. We applied our approach in a phenotypic screening workflow and found novel nanomolar inhibitors of cell division that reproduce the mode of action of reference natural products. In a drug discovery setting, our biological hit expansion protocol revealed new inhibitors of the NKCC1 co-transporter for autism spectrum disorders. Furthermore, we demonstrate BIOSEA's capabilities to predict novel targets for old compounds. We report new activities for the drugs nimedipine, fluspirilene and pimozide applicable for compound repurposing and rationalizing drug side effects. Our results highlight the opportunities of reusing public bioactivity data for prospective drug discovery applications where the target or mode of action is not known.

Maire F. Osborn, Andrew H Coles, Annabelle Biscans, Reka A Haraszti, Loic Roux, Sarah Davis, Socheata Ly, Dimas Echeverria, Matthew R Hassler, Bruno M.D.C. Godinho, Mehran Nikan, Anastasia Khvorova

Efficient delivery of therapeutic RNA is the fundamental obstacle preventing its clinical utility. Lipid conjugation improves plasma half-life, tissue accumulation, and cellular uptake of small interfering RNAs (siRNAs). However, the impact of conjugate structure and hydrophobicity on siRNA pharmacokinetics is unclear, impeding the design of clinically relevant lipid-siRNAs. Using a panel of biologically-occurring lipids, we show that lipid conjugation modulates siRNA hydrophobicity and governs spontaneous partitioning into distinct plasma lipoprotein classes in vivo. Lipoprotein binding influences siRNA distribution by delaying renal excretion and promoting uptake into lipoprotein receptor-enriched tissues. Lipid-siRNAs elicit mRNA silencing without causing toxicity in a tissue-specific manner. Lipid-siRNA internalization occurs independently of lipoprotein endocytosis, and is mediated by siRNA phosphorothioate modifications. Although biomimetic lipoprotein nanoparticles have been considered for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

Mateusz Maciejewski, Eugen Lounkine, Steven Whitebread, Pierre Farmer, Bill DuMouchel, Brian K. Shoichet, Laszlo Urban

The Food and Drug Administration Adverse Event Reporting System (FAERS) is the primary source for postmarketing pharmacovigilance. Though potentially highly useful, the database reflects reporting biases, stimulated reporting, and suffers from lack of standardization and the use of multiple drug synonyms. These biases can suggest adverse drug reactions (ADRs) where none exist, and can obscure others that do exist. To decrease the noise in FAERS, and to reinforce important associations, we mapped over 750,000 drug identifiers in FAERS to the normalized chemical structures of their ingredients. This illuminated associations that would not otherwise be apparent, and also allowed a time-resolved analysis of ADR reporting. It also revealed similarities between drugs and adverse events across therapeutic classes, enabling unbiased classification of adverse events, indications, and drugs with similar clinical profiles. For instance, comparison of two selective cyclooxygenase-2 inhibitors, celecoxib and rofecoxib finds distinctive FAERS profiles after time-resolved analysis. We also investigated key idiosyncrasies, such as confusion between drug indications and drug ADRs, which can tar a drug treating a life-threatening disease, like thalidomide's use against myeloma, with a deadly ADR that is likely the result of the disease itself, multiplications of the same report, which unjustifiably increases its apparent importance, and the correlation of reported ADRs with public events, regulatory announcements, and with publications. Comparing the pharmacological, pharmacokinetic, and clinical ADR profiles of methylphenidate, aripiprazole and risperidone, and of kinase drugs targeting the VEGF receptor (VEGF-R2), demonstrates how underlying molecular mechanisms can emerge from ADR co-analysis. The precautions and methods we describe may enable investigators to avoid confounding chemistry-based associations and reporting biases in FAERS, and illustrate how comparative analysis of ADRs can reveal underlaying mechanisms.

Robin Mesnage, Alexia Phedonos, Matthew Arno, Sucharitha Balu, J. Christopher Corton, Michael N Antoniou

Background: Plasticizers with estrogenic activity, such as bisphenol A (BPA), have been reported to have potential adverse health effects in humans. Due to mounting evidence of these health effects and public pressure, BPA is being phased out by the plastics manufacturing industry and replaced by other bisphenol variants in 'BPA-free' products. Objectives: We have compared estrogenic activity of BPA to 6 bisphenol analogues (bisphenol S, BPS; bisphenol F, BPF; bisphenol AP, BPAP; bisphenol AF, BPAF; bisphenol Z, BPZ; bisphenol B, BPB) in three human breast cancer cell lines. Methods: Estrogenicity was assessed by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element (ERE)-mediated transcription in a luciferase assay. Gene expression profiles were determined in MCF-7 human breast cancer cells by microarray analysis and confirmed by Illumina-based RNA sequencing. Results: All bisphenols showed estrogenic activity in promoting cell growth and inducing ERE-mediated transcription. BPAF was the most potent bisphenol, followed by BPB > BPZ ~ BPA > BPF ~ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs by bisphenols. Data mining of ToxCast high-throughput screening assays confirms our results but also shows divergence in the sensitivities of the assays. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα; gene expression biomarker further indicates that all BPA alternatives act as ERα; agonists in MCF-7 cells. These results were confirmed by RNA sequencing. Conclusion: In conclusion, BPA alternatives are not necessarily less estrogenic in a human breast cancer cell model. Three bisphenols (BPAF, BPB, and BPZ) were more estrogenic than BPA. The relevance of human exposure to BPA alternatives in hormone-dependent breast cancer risk should be investigated.

Guilherme Duarte Ramos Matos, Daisy Y. Kyu, Hannes H. Loeffler, John D. Chodera, Michael R. Shirts, David L. Mobley

Solvation free energies can now be calculated precisely from molecular simulations, providing a valuable test of the energy functions underlying these simulations. Here, we briefly review "alchemical" approaches for calculating the solvation free energies of small, neutral organic molecules from molecular simulations, and illustrate by applying them to calculate aqueous solvation free energies (hydration free energies). These approaches use a non-physical pathway to compute free energy differences from a simulation or set of simulations and appear to be a particularly robust and general-purpose approach for this task. We also present an update (version 0.5) to our FreeSolv database of experimental and calculated hydration free energies of neutral compounds and provide input files in formats for several simulation packages. This revision to FreeSolv provides calculated values generated with a single protocol and software version, rather than the heterogeneous protocols used in the prior version of the database. We also further update the database to provide calculated enthalpies and entropies of hydration and some experimental enthalpies and entropies, as well as electrostatic and nonpolar components of solvation free energies.

Marina Santiago, Shivani Sachdev, Jonathon C Arnold, Iain S McGregor, Mark Connor

Introduction: Compounds present in Cannabis sativa such as phytocannabinoids and terpenoids, may act in concert to elicit therapeutic effects. Cannabinoids such as Δ9-tetrahydrocannabinol (Δ9-THC) directly activate cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), however, it is not known if terpenoids present in Cannabis also affect cannabinoid receptor signalling. Therefore, we examined 6 common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB1 and CB2 signalling in vitro. Materials and Methods: Potassium channel activity in AtT20 FlpIn cells transfected with human CB1 or CB2 receptors was measured in real-time using FLIPR membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30 minutes. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels. Results: α-Pinene, β-pinene, β-caryophyllene, linalool, limonene and β-myrcene (up to 30-100 μM) did not change membrane potential in AtT20 cells expressing CB1 or CB2, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by Δ9-THC (10μM): (CB1: control, 59 ± 7%; with terpenoids (10 μM each) 55 ± 4%; CB2: Δ9-THC 16±5%, with terpenoids (10 μM each) 17±4%). To investigate possible effect on desensitization of CB1 responses, all six terpenoids were added together with Δ9-THC and signalling measured continuously over 30 min. Terpenoids did not affect desensitization, after 30 minutes the control hyperpolarization recovered by 63±6%, in the presence of the terpenoids recovery was 61±5%. Discussion: None of the six of the most common terpenoids in Cannabis directly activated CB1 or CB2, or modulated the signalling of the phytocannabinoid agonist Δ9-THC. These results suggest that if a phytocannabinoid-terpenoid entourage effect exists, it is not at the CB1 or CB2 receptor level. It remains possible that terpenoids activate CB1 and CB2 signalling pathways that do not involve potassium channels, however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioural effect of Cannabis.