Esther E. 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.

N.V Rajeshkumar, Shinichi Yabuuchi, Shweta G Pai, Anirban Maitra, Manuel Hidalgo, Chi V Dang

Guided by the principle of primum non nocere (first do no harm), we report a cautionary note on the potential fatal toxicity of chloroquine (CQ) or hydroxychloroquine (HCQ) in combination with anti-diabetic drug metformin. We observed that the combination of CQ or HCQ and metformin, which were used in our studies as potential anti-cancer drugs, killed 30-40% of mice. While our observations in mice may not translate to toxicity in humans, the reports that CQ or HCQ has anti-COVID-19 activity, the use of CQ resulting in toxicity and at least one death, and the recent Emergency Use Authorization (EUA) for CQ and HCQ by the US Food and Drug Administration (FDA) prompted our report. Here we report the lethality of CQ or HCQ in combination with metformin as a warning of its potential serious clinical toxicity. We hope that our report will be helpful to stimulate pharmacovigilance and monitoring of adverse drug reactions with the use of CQ or HCQ, particularly in combination with metformin.

Zhijian Xu, Cheng Peng, Yulong Shi, Zhengdan Zhu, Kaijie Mu, Xiaoyu Wang, Weiliang Zhu

2019-nCov has caused more than 80 deaths as of 27 January 2020 in China, and infection cases have been reported in more than 10 countries. However, there is no approved drug to treat the disease. 2019-nCov Mpro is a potential drug target to combat the virus. We built homology models based on SARS Mpro structures, and docked 1903 small molecule drugs to the models. Based on the docking score and the 3D similarity of the binding mode to the known Mpro ligands, 4 drugs were selected for binding free energy calculations. Both MM/GBSA and SIE methods voted for nelfinavir, with the binding free energy of -24.69±0.52 kcal/mol and -9.42±0.04 kcal/mol, respectively. Therefore, we suggested that nelfinavir might be a potential inhibitor against 2019-nCov Mpro.

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.

Yan Li, Jinyong Zhang, Ning Wang, Haibo Li, Yun Shi, Gang Guo, Kaiyun Liu, Hao Zeng, Quanming Zou

2019 Novel Coronavirus (2019-nCoV) is a virus identified as the cause of the outbreak of pneumonia first detected in Wuhan, China. Investigations on the transmissibility, severity, and other features associated with this virus are ongoing. Currently, there is no vaccine or therapeutic antibody to prevent the infection, and more time is required to develop an effective immune strategy against the pathogen. In contrast, specific inhibitors targeting the key protease involved in replication and proliferation of the virus are the most effective means to alleviate the epidemic. The main protease of SARS-CoV is essential for the life cycle of the virus, which showed 96.1% of similarity with the main proteaseof 2019-nCoV, is considered to be an attractive target for drug development. In this study, we have identified 4 small molecular drugs with high binding capacity with SARS-CoV main protease by high-throughput screening based on the 8,000 clinical drug libraries, all these drugs have been widely used in clinical applications with guaranteed safety, which may serve as promising candidates to treat the infection of 2019-nCoV.

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

Bowen Tang, Fengming He, Dongpeng Liu, Meijuan Fang, Zhen Wu, Dong Xu

The focused drug repurposing of known approved drugs (such as lopinavir/ritonavir) has been reported failed for curing SARS-CoV-2 infected patients. It is urgent to generate new chemical entities against this virus. As a key enzyme in the life-cycle of coronavirus, the 3C-like main protease (3CLpro or Mpro) is the most attractive for antiviral drug design. Based on a recently solved structure (PDB ID: 6LU7), we developed a novel advanced deep Q-learning network with the fragment-based drug design (ADQN-FBDD) for generating potential lead compounds targeting SARS-CoV-2 3CLpro. We obtained a series of derivatives from those lead compounds by our structure-based optimization policy (SBOP). All the 47 lead compounds directly from our AI-model and related derivatives based on SBOP are accessible in our molecular library at https://github.com/tbwxmu/2019-nCov. These compounds can be used as potential candidates for researchers in their development of drugs against SARS-CoV-2.

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.

Jingyue Ju, Shiv Kumar, Xiaoxu Li, Steffen Jockusch, James J. Russo

Coronaviruses such as the newly discovered virus from Wuhan, China, 2019-nCoV, and the viruses that cause SARS and MERS, have resulted in regional and global public health emergencies. Based on our molecular insight that the hepatitis C virus and the coronavirus use a similar viral genome replication mechanism, we reasoned that the FDA-approved drug EPCLUSA (Sofosbuvir/Velpatasvir) for the treatment of hepatitis C will also inhibit the above coronaviruses, including 2019-nCoV. To develop broad spectrum anti-viral agents, we further describe a novel strategy to design and synthesize viral polymerase inhibitors, by combining the ProTide Prodrug approach used in the development of Sofosbuvir with the use of 3′-blocking groups that we have previously built into nucleotide analogues that function as polymerase terminators.

Jingyue Ju, Xiaoxu Li, Shiv Kumar, Steffen Jockusch, Minchen Chien, Chuanjuan Tao, Irina Morozova, Sergey Kalachikov, Robert N. Kirchdoerfer, James J. Russo

SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved heptatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the activated triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the activated triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected two other anti-viral agents, Alovudine and AZT (an FDA approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two HIV reverse transcriptase inhibitors, 3′-fluoro-3′-deoxythymidine triphosphate and 3′-azido-3′-deoxythymidine triphosphate (the active triphosphate forms of Alovudine and AZT), to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.

Steffen Jockusch, Chuanjuan Tao, Xiaoxu Li, Thomas K. Anderson, Minchen Chien, Shiv Kumar, James J. Russo, Robert N. Kirchdoerfer, Jingyue Ju

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. We previously demonstrated that four nucleotide analogues (specifically, the active triphosphate forms of Sofosbuvir, Alovudine, AZT and Tenofovir alafenamide) inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Tenofovir and emtricitabine are the two components in DESCOVY and TRUVADA, the two FDA-approved medications for use as pre-exposure prophylaxis (PrEP) to prevent HIV infection. This is a preventative method in which individuals who are HIV negative (but at high-risk of contracting the virus) take the combination drug daily to reduce the chance of becoming infected with HIV. PrEP can stop HIV from replicating and spreading throughout the body. We report here that the triphosphates of tenofovir and emtricitabine, the two components in DESCOVY and TRUVADA, act as terminators for the SARS-CoV-2 RdRp catalyzed reaction. These results provide a molecular basis to evaluate the potential of DESCOVY and TRUVADA as PrEP for COVID-19.

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.

Hongbo Liu, Fei Ye, Qi Sun, Hao Liang, Chunmei Li, Roujian Lu, Baoying Huang, Wenjie Tan, Luhua Lai

COVID-19 has become a global pandemic that threatens millions of people worldwide. There is an urgent call for developing effective drugs against the virus (SARS-CoV-2) causing this disease. The main protease of SARS-CoV-2, 3C-like protease (3CLpro), is highly conserved across coronaviruses and is essential for the maturation process of viral polyprotein. Scutellariae radix (Huangqin in Chinese), the root of Scutellaria baicalensis has been widely used in traditional Chinese medicine to treat viral infection related symptoms. The extracts of S. baicalensis have exhibited broad spectrum antiviral activities. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredient compounds. We found that the ethanol extract of S. baicalensis inhibits SARS-CoV-2 3CLpro activity in vitro and the replication of SARS-CoV-2 in Vero cells with an EC50 of 0.74 μg/ml. Among the major components of S. baicalensis, baicalein strongly inhibits SARS-CoV-2 3CLpro activity with an IC50 of 0.39 μM. We further identified four baicalein analogue compounds from other herbs that inhibit SARS-CoV-2 3CLpro activity at microM concentration. Our study demonstrates that the extract of S. baicalensis has effective anti-SARS-CoV-2 activity and baicalein and analogue compounds are strong SARS-CoV-2 3CLpro inhibitors. ### Competing Interest Statement The authors have declared no competing interest.

Minchen Chien, Thomas K. Anderson, Steffen Jockusch, Chuanjuan Tao, Shiv Kumar, Xiaoxu Li, James J. Russo, Robert N. Kirchdoerfer, Jingyue Ju

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). Here, using polymerase extension experiments, we have demonstrated that the active triphosphate form of Sofosbuvir (a key component of the FDA approved hepatitis C drug EPCLUSA), is incorporated by SARS-CoV-2 RdRp, and blocks further incorporation. Using the same molecular insight, we selected the active triphosphate forms of three other anti-viral agents, Alovudine, AZT (an FDA approved HIV/AIDS drug) and Tenofovir alafenamide (TAF, an FDA approved drug for HIV and hepatitis B) for evaluation as inhibitors of SARS-CoV-2 RdRp. We demonstrated the ability of these three viral polymerase inhibitors, 3′-fluoro-3′-deoxythymidine triphosphate, 3′-azido-3′-deoxythymidine triphosphate and Tenofovir diphosphate (the active triphosphate forms of Alovudine, AZT and TAF, respectively) to be incorporated by SARS-CoV-2 RdRp, where they also terminate further polymerase extension. These results offer a strong molecular basis for these nucleotide analogues to be evaluated as potential therapeutics for COVID-19.

Chunlong Ma, Michael D. Sacco, Brett Hurst, Julia A Townsend, Yanmei Hu, Tommy Szeto, Xiujun Zhang, Bart Tarbet, Michael T Marty, Yu Chen, Jun Wang

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 μM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics. ### Competing Interest Statement J. W. and C. M. are inventors of a pending patent that claims the use of the identified compounds for COVID-19.

Wen-Hao Guo, Xiaoli Qi, Xin Yu, Yang Liu, Chan-I Chung, Fang Bai, Xingcheng Lin, Dong Lu, Lingfei Wang, Jianwei Chen, Lynn Su, Krystle J. Nomie, Ashok Litwin-Kumar, Meng C. Wang, Xiaokun Shu, Jose N. Onuchic, Jennifer A. Woyach, Michael L. Wang, Jin Wang

Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton Tyrosine Kinase (BTK), we serendipitously discovered that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and developed RC 1 as the first reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor/degrader, a novel mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a new path to enhance PROTAC efficacy. ### Competing Interest Statement J.W., W.G., X.Q., M.L.W., K.N., and Y.L. are the co-inventors of a patent related to this work. J.W. is the co-founder of CoActigon Inc. and Chemical Biology Probes LLC. X.S. and C.-I.C. have filed a patent application covering the SPPIER assay. M.L.W. receives research grants from Janssen, Pharmacyclics, AstraZeneca, Acerta Pharma, Celgene, Juno Therapeutics, BeiGene, Kite Pharma, Loxo Oncology, VelosBio, BioInvent, Aviara, Verastem, and InnoCare, consults and/or serves on the advisory board for Consulting/Advisory Board for Loxo Oncology, Janssen, Pharmacyclics, BioInvent, Celgene, Juno Therapeutics, Pulse Biosciences, MoreHealth, Guidepoint Global, Kite Pharma, AstraZeneca, Acerta Pharma, and InnoCare, received honoraria from Janssen, Acerta Pharma, OMI, Physicians Education Resources (PER), Oncology News, and Targeted Oncology, and owns stocks from MoreHealth. J.A.W. consults for Janssen, Pharmacyclics, Abbvie, AstraZeneca, and Arqule, receives research funding from Loxo Oncology and Abbvie and clinical trial funding from Pharmacyclics, Janssen, Verastem, Karyopharm, and Morphosys.

L Miyashita, G Foley, S Semple, J Grigg

Background: The mechanism for the association between traffic-derived particulate matter less than 10 microns (PM10) and cases of COVID-19 disease reported in epidemiological studies is unknown. To infect cells, the spike protein of SARS-CoV-2 interacts with angiotensin-converting enzyme 2 (ACE2) on host airway cells. Increased ACE2 expression in lower airway cells in active smokers, suggests a potential mechanism whereby PM10 increases vulnerability to COVID-19 disease. Objective: To assess the effect of traffic-derived PM10 on human airway epithelial cell ACE2 expression in vitro. Methods: PM10 was collected from Marylebone Road (London) using a kerbside impactor. A549 and human primary nasal epithelial cells were cultured with PM10 for 2 h, and ACE2 expression (median fluorescent intensity; MFI) assessed by flow cytometry. We included cigarette smoke extract as a putative positive control. Data were analysed by either Mann-Whitney test, or Kruskal-Wallis with multiple comparisons test. Results: PM10 at 10 μg/mL, and 20 μg/mL increased ACE2 expression in A549 cells (P<0.05, 0.01 vs. medium control, respectively). Experiments using a single PM10 concentration (10 μg/mL), found increased ACE2 expression in both A549 cells (control vs. PM10, median (IQR) MFI; 470 (0.1 to 1114) vs 6217 (5071 to 8506), P<0.01), and in human primary epithelial cells (0 (0 to 591) vs. 4000 (2610 to 7853), P<0.05). Culture of A549 cells with 5% cigarette smoke extract increased ACE2 expression (n=4, 0 (0 to 28) vs. 9088 (7557 to 15831, P<0.05). Conclusion: Traffic-related PM10 increases the expression of the receptor for SARS-CoV-2 in human respiratory epithelial cells. ### Competing Interest Statement The authors have declared no competing interest.

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.

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.

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.