Screening Of Molecules Research Articles

Development of a cells and bead-based, advanced flow cytometry assay for exhausted T cell quantitation

Abstract T cells can become exhausted due to persistent antigen exposure during chronic infections and cancer. Exhausted T cells exhibit decreased effector functions and express inhibitory receptors (IRs). The exhausted phenotype may be protective in the context of chronic infection, however, it allows tumors to avoid immune surveillance and immune cell mediated killing. Recent therapies block the IRs to reinvigorate exhausted T cells. A miniaturized, multiplexed flow cytometry assay was developed to measure expression of IRs on T cells while simultaneously measuring viability, proliferation, and cytokine secretion in microtiter plates. Data was acquired on the Intellicyt® iQue platform and analyzed using Forecyt®. Templates were prepared for ease of data analysis, to auto-generate gating, standard curves and sample quantitation and rapid, actionable results. For proof of concept studies, human PBMCs, purified CD3+, or in vitro exhausted purified CD3+ cells were treated with anti-CD3/CD28 and cells were analyzed at 1, 2, and 3 days post-stimulation. Non-exhausted cells showed a concentration and time dependent increase in IR expression. Persistent, high levels of the IR markers PD-1, LAG-3 and TIM-3 were measured in exhausted cells. Stimulated exhausted T cells secreted stable and moderate levels of IFN-γ (<1650 pg/ml) and TNF (<750 pg/ml) over time whereas non-exhausted cells showed time and concentration dependent increases in IFN-γ (13,319 pg/ml) and TNF (3,711 pg/ml) upon stimulation. The ability of this assay to examine T cell response allows screening for molecules that reverse T cell exhaustion, screening of modified cells such as CAR-Ts or selection of clones that exhibit low levels of IRs and show robust response to stimulation.

Probing the Ultrastructure of Spheroids and Their Uptake of Magnetic Nanoparticles by FIB–SEM

AbstractSpheroids are 3D cellular systems largely adopted as model for high‐throughput screening of molecules and diagnostics tools. Furthermore, those cellular platforms also represent a model for testing new delivery carries for selective targeting. The coupling between the 3D cell environment and the nanovectors can be explored at the macroscale by optical microscopy. However, the nanomaterial‐cell interplay finds major action at the single cell and extracellular matrix level with nanoscale interactions. Electron microscopy offers the resolution to investigate those interactions; however, the specimen preparation finds major drawbacks in its operation time and preciseness. In this context, focused ion beam and scanning electron microscopy (FIB–SEM) allows for fast processing and high resolution of the cell‐nanomaterial interface. Here, in fact, a novel approach is shown to prepare large‐area 3D spheroid cell culture specimens for FIB–SEM. Sectioning procedures are explored to preserve the peculiar structure of spheroids and their interaction with magnetic nanovectors. The results pave the way for advanced investigations of 3D cellular systems with nano and micromaterials relevant to tissue engineering, bioelectronics, and diagnostics.

Structure-based virtual screening, free energy of binding and molecular dynamics simulations to propose novel inhibitors of Mtb-MurB oxidoreductase enzyme

Currently, the growing incidence of drug resistance toward tuberculosis intensified the need for discovery of novel targets and their inhibitors. The enzyme MurB which is involved in one of the steps for peptidoglycan biosynthesis is an effective target that can produce drugs having lesser side-effects. Recently the only crystal structure of Mycobacterium Tuberculosis MurB has been deposited and, therefore, in the present study, we have used this as a target for virtual screening of drug-like molecules from the ZINC Database. We have also designed a complete workflow for the process which resulted in 12 hit compounds that have good docking scores, ΔGbind, and Glide energy. The hits obtained have also been found to share structural features with some known antibiotics such as Amoxicillin. Furthermore, MD simulations on the top most hit L1 displayed its stable binding with the enzyme. Thus, this study has proved helpful in proposing novel inhibitors for MurB enzyme that can be tested against various TB strains.

Electrodynamic characteristics of λ-DNA molecule translocating through the microfluidic channel port studied with single molecular fluorescence imaging technology

Manipulating a single DNA molecule and effectively introducing it into and exporting micro-nano-fluidic channels are prerequisites for the functional DNA biochips. And it is the key to the precise separation and screening of different DNA molecules by the micro-/nanochannel system that accurately understanding the movement characteristics and dynamic mechanism of DNA molecules moving near the channel port. In this paper, the electrodynamic characteristics of λ-DNA molecule entering into/leaving off a 50 μm channel port driven by the electric field force are systematically investigated and analyzed by the single molecule fluorescence microscopy. The experimental results indicated that there were the maximum (<i>E</i><sub>max</sub>) and minimum (<i>E</i><sub>min</sub>) thresholds of the applied electric field intensity, and only when the field intensity <i>E</i> meets <i>E</i><sub>min</sub> ≤ <i>E</i> ≤ <i>E</i><sub>max</sub>, the single λ-DNA molecule could successfully enter into the <i>trans</i> port and exit out of the <i>cis</i> port; when the electric field intensity was less than the minimum threshold, <i>E</i> ≤ <i>E</i><sub>min</sub>, λ-DNA molecules could not enter the <i>trans</i> port; when the electric field intensity was greater than the maximum threshold, <i>E</i><sub>max</sub> ≤ <i>E</i>, λ-DNA molecules could move into the microchannel through the <i>trans</i> port, but not exit out of the <i>cis</i> port. When λ-DNA molecule migrated toward the <i>cis</i> port along the channel, the movement state was changed, some new phenomena were observed, e.g. the translocation direction was reversed, reciprocated, or even rotated; moreover, the DNA molecules were easy to adhere to the channel wall. In addition, when the electric field intensity enhanced, the distance between the position where DNA molecular direction reversing and the <i>cis</i> port was increased. Based on the microfluidic electrodynamics, the physical mechanism of the velocities and translocation states of single λ-DNA molecule passing microchannel port was preliminarily analyzed. The results of this study have certain practical guiding significance for the development of gene chip laboratory and DNA molecular sensors based on the micro/nanochannel fluidic system.

Screening of promising molecules against MurG as drug target in multi-drug-resistant-Acinetobacter baumannii - insights from comparative protein modeling, molecular docking and molecular dynamics simulation

The UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) is located in plasma membrane which plays a crucial role for peptidoglycan biosynthesis in Gram-negative bacteria. Recently, this protein is considered as an important and unique drug target in Acinetobacter baumannii since it plays a key role during the synthesis of peptidoglycan as well as which is not found in Homo sapiens. In this study, initially we performed comparative protein modeling approach to predict the three-dimensional model of MurG based on crystal structure of UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (PDB ID: 1F0K) from E.coli K12. MurG model has two important functional domains located in N and C- terminus which are separated by a deep cleft. Active site residues are located between two domains and they are Gly20, Arg170, Gly200, Ser201, Gln227, Phe254, Leu275, Thr276, and Glu279 which play essential role for the function of MurG. In order to inhibit the function of MurG, we employed the High Throughput Virtual Screening (HTVS) and docking techniques to identify the promising molecules which will further subjected into screening for computing their drug like and pharmacokinetic properties. From the HTVS, we identified 5279 molecules, among these, 12 were passed the drug-like and pharmacokinetic screening analysis. Based on the interaction analysis in terms of binding affinity, inhibition constant and intermolecular interactions, we selected four molecules for further MD simulation to understand the structural stability of protein-ligand complexes. All the analysis of MD simulation suggested that ZINC09186673 and ZINC09956120 are identified as most promising putative inhibitors for MurG protein in A. baumannii.Communicated by Ramaswamy H. Sarma

A multiple classifier system identifies novel cannabinoid CB2 receptor ligands

Drugs have become an essential part of our lives due to their ability to improve people’s health and quality of life. However, for many diseases, approved drugs are not yet available or existing drugs have undesirable side effects, making the pharmaceutical industry strive to discover new drugs and active compounds. The development of drugs is an expensive process, which typically starts with the detection of candidate molecules (screening) after a protein target has been identified. To this end, the use of high-performance screening techniques has become a critical issue in order to palliate the high costs. Therefore, the popularity of computer-based screening (often called virtual screening or in silico screening) has rapidly increased during the last decade. A wide variety of Machine Learning (ML) techniques has been used in conjunction with chemical structure and physicochemical properties for screening purposes including (i) simple classifiers, (ii) ensemble methods, and more recently (iii) Multiple Classifier Systems (MCS). Here, we apply an MCS for virtual screening (D2-MCS) using circular fingerprints. We applied our technique to a dataset of cannabinoid CB2 ligands obtained from the ChEMBL database. The HTS collection of Enamine (1,834,362 compounds), was virtually screened to identify 48,232 potential active molecules using D2-MCS. Identified molecules were ranked to select 21 promising novel compounds for in vitro evaluation. Experimental validation confirmed six highly active hits (> 50% displacement at 10 µM and subsequent Ki determination) and an additional five medium active hits (> 25% displacement at 10 µM). Hence, D2-MCS provided a hit rate of 29% for highly active compounds and an overall hit rate of 52%.

In silico screening of drug-like molecules for the treatment of cardiovascular diseases on the basis of five-membered privileged heterocycles

Among various heterocyclic systems, the derivatives of five-membered heterocycles are of special interest. Most of the above mentioned heterocycles are treatred as so-called privileged structures in modern medicinal chemistry. In silico screening among five-membered heterocycles of molecules for the treatment of cardiovascular diseases is actual.
 The aim of the work was the search for synthetic drug-like molecules based on functionalized five-membered heterocycles and related heterocyclic systems as an element of the theoretical platform for rational design of compounds acting on the cardiovascular system, and prediction of their possible mechanism of action.
 The objects of the study were derivatives of uncondensed and condensed five-membered heterocycles. In the work, in silico approaches were applied using the programs: Hyper-Chem, PASS, AutoDock, PROTOX.
 Based on the previous studies, focused sub-libraries of small synthetic drug-like molecules based on functionalized five-membered heterocycles and related heterocyclic systems have been selected. The compounds were divided on 12 groups. The optimization of the compound structures, the drug-like parameters calculation were carried out. The activity prediction, the acute toxicity level and docking studies to probable bio-targets which are related with cardiovascular drug mechanism of action have been carried out. It was shown that thiazole and thiadiazole based compounds possessed the highest calculated affinity levels to selected bio-targets. This is consistent with PASS-based prediction data.
 Diverse functionalized derivatives of five-membered heterocycles (thiazole, thiazolidine, thiadiazole, pyrazole, thiophene, triazole) and related fused heterocycles have been grouped in focused sub-libraries of compounds. it has been established that thiazole and thiadiazole based compounds are promising objects for directed synthesis and further modification as potential cardiovascular agents based on the prediction of biological activity, the calculation of affinity to potent bio-targets, and the prediction of the drug-like features and acute toxicity level. The prognostic values of the parameters of the above mentioned groups of compounds may be used as the element of theoretical platform for the search and de novo design of potential drugs for the treatment of cardiovascular diseases.

Abstract 2690: Massively parallel multiplexed methods to screen hundreds of barcoded cancer cell line models with small molecules or genetic perturbations using next-generation sequencing

Abstract Phenotypic screening is a valuable tool to identify compounds to treat cancer, but is limited as it is time and resource intensive to screen hundreds of cancer cell lines. In order to increase the throughput of phenotypic screening, we set out to expand and further develop the PRISM method (Profiling Relative Inhibition Simultaneously in Mixtures) [Yu et al., 2016]. PRISM is a method to barcode cell lines with a unique 24 nucleotide barcode and mix them together to screen simultaneously in a pool, decreasing the time and cost of screening. The previously described PRISM method was limited in that it used only 100 adherent cell lines of one cancer model context, it required the use of a highly specialized Luminex bead-based detection system, and it was only applicable to small molecule screening. Here we report on the expansion of our barcoded cell line collection to 800 cancer cell lines of over 45 lineages, an improved method called PRISMseq using next-generation sequencing, and a novel method to perform genetic perturbations in 500 cancer cell lines simultaneously called PRISPR (PRISM/CRISPR). PRISMseq allows for assaying compound cell line sensitivity profiles in a large pool of hundreds of cell lines and detecting the DNA barcodes using next-generation sequencing (NGS). We recapitulate the known biology for established oncology drugs like the BRAF, EGFR, BCR-ABL and MDM2 inhibitor classes using this method with our cell line panel. We also further extended this method to be applicable to genetic perturbation using CRISPR/Cas9 knockout of individual genes concurrently in hundreds of cancer cell lines. We are able to recover expected biomarkers or genetic dependencies for our CRISPR/Cas9 validation reagents. This expanded PRISM profiling approach increases statistical power due to the addition of cancer contexts in our cell line collection, and improves versatility by enabling the screening of both small molecules and genetic perturbations. We believe that this method has overcome the limitations of the original PRISM method. It has also overcome the limitations of phenotypic screening, as the resources required to screen hundreds of cell lines has been decreased by orders of magnitude. Citation Format: Samantha Bender, Cong Zhu, Li Wang, Michael Rothberg, Joshua Dempster, Brenton Paolella, Mustafa Kocak, Massami Laird, Jordan Rossen, Karolina Stumbraite, Jordan Bryan, Vickie Wang, John Doench, Francisca Vazquez, Aviad Tsherniak, Todd Golub, Jennifer Roth. Massively parallel multiplexed methods to screen hundreds of barcoded cancer cell line models with small molecules or genetic perturbations using next-generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2690.

Screening of rice accessions and newer molecules against lepidopterous pests in Manipur, India

Trials conducted to assess the resistant reaction of some rice accessions and evaluation of newer molecules against the incidence of two key lepidopterous pests viz., Scirpophaga incertulas Walker and Cnaphalocrocis medinalis Guenee revealed that among the screened varieties, WR-15-6-1, WR-1-9-1-1, CAU-R2, Lamyanbaphou, RC-MANI-PHOU-11, Matamphou, KD-6-18-7-1 and RCMANIPHOU-6-RC-5 reacted moderately susceptible at vegetative stage and among these eight, four varieties viz., WR-1-9-1-1, CAU-R2, RC-MANI-PHOU-11 and Matamphou showed resistant reaction at heading stage against S. incertulas and six accessions viz., WR-1-9-1-1, CAU-R3, KD-5-3-14, RCMANI-PHOU-11, CAU-R4 and KD-6-18-7-1 were categorized as moderately susceptible to C. medinalis. The test on efficacy of newer molecule showed that treatment with Fipronil 5 SC @ 100 ml a.i. ha1 was found to be most effective in reducing Deadheart (DH) and White Ear Head (WEH) with minimum incidence of 3.85 per cent and 1.87% WEH respectively. Whereas the pooled mean data of two spray indicated that Imidacloprid 17.8 SL @ 250 ml a.i. ha1 showed minimum leaf damage of 4.16 % which was at par with Flubendiamide 39.35 SC @ 50 g a.i. ha-1 (4.63% LD), Fipronil 5 SC @ 100 ml a.i. ha-1.

Enhanced competitive immunomagnetic beads assay with Gold Nanoparticles and Carbon Nanotube-Assisted Multiple enzyme probes

Various nanoparticles provide new nanoscaffolds for enzymes to immobilize and develop a series of multi-enzyme probes for sensitive assays. In this experiment, gold nanoparticles (AuNPs) and multi-well carbon nanotubes (MWCNTs) were adopted as solid support and different conjugation methods with horseradish peroxidase (HRP) were explored. Kanamycin (KAN), one of amino glycoside antibiotics, was adopted as the target to develop the enhanced MPs-based competitive immunoassays. Three multi-enzyme probes, Au/KAN-HRP/HRP, MWCNTs/KAN-HRP/HRP-p and MWCNTs/KAN-HRP/HRP-c, were synthesized. MWCNTs/KAN-HRP/HRP-p showed highest catalytic efficiency, following MWCNTs/KAN-HRP/HRP-c, and Au/KAN-HRP/HRP lowest. With the optimal condition, enhanced competitive MPs immunoassays with these multi-enzyme probes were established to detect KAN. The value of LOD were 128 pg/mL, 17 pg/mL and 6.99 pg/mL based on Au/KAN-HRP/HRP, MWCNTs/KAN-HRP/HRP-c and MWCNTs/KAN-HRP/HRP-p respectively, which improved 2-fold, 15-fold and 37-fold respectively compared with that of conventional IMBs assay. With the aid of multi-enzyme probes based on nanoparticles, it is promising for sensitive detection and screening of molecules from biological samples.

Screening of Natural Lead Molecules Against Putative Molecular Targets of Drug-resistant Cryptococcus spp: An Insight from Computer-aided Molecular Design.

Cryptococcosis is one of the major invasive fungal infections distributed worldwide with high mortality rate. C. neoformans and C. gattii are the major organisms that cause various types of infections. Anti-fungal resistances exhibited by the mentioned species of Cryptococcus threaten their effective prevention and treatment. There is limited information available on human to human transmission of the pathogen and virulent factors that are responsible for Cryptococcus mediated infections. Hence, there is high scope for understanding the mechanism, probable drug targets and scope of developing natural therapeutic agents that possess high relevance to pharmaceutical biotechnology and medicinal chemistry. The proposed review illustrates the role of computer-aided virtual screening for the screening of probable drug targets and identification of natural lead candidates as therapeutic remedies. The review initially focuses on the current perspectives on cryptococcosis, major metabolic pathways responsible for the pathogenesis, conventional therapies and associated drug resistance, challenges and scope of structure-based drug discovery. The review further illustrates various approaches for the prediction of unknown drug targets, molecular modeling works, screening of natural compounds by computational virtual screening with ideal drug likeliness and pharmacokinetic features, application of molecular docking studies and simulation. Thus, the present review probably provides AN insight into the role of medicinal chemistry and computational drug discovery to combat Cryptococcus infections and thereby open a new paradigm for the development of novel natural therapeutic against various drug targets for cryptococcal infections.

Microfluidic Device for Screening for Target Cell-Specific Binding Molecules by Using Adherent Cells.

This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on the surface of target cells, washing away unbound molecules, and collecting target cell-specific binding molecules from target cells. These functions were effectively implemented by using our previously developed micro pillar arrays for cell homogeneous dispersion and pneumatic microvalves for tall microchannels. The device was also equipped with serially connected filter chambers in which non-target cells were cultured to reduce the molecules binding to non-target cells as much as possible. We evaluated the performance of the device using cancer cell lines (N87 cells as target cells and HeLa cells as non-target cells) and two fluorescent dye-labeled antibodies: Anti-human epidermal growth factor receptor 2 (anti-HER2) antibody that binds to target cells and anti-integrin antibody that binds to non-target cells. The results showed that the device could reduce anti-integrin antibodies to the detection limit of fluorescent measurement and collect anti-HER2 antibodies from the target cells.

Probing of Phytofungal Proteins for Fungicidal Activity by Molecular Docking

Background: Plant fungal diseases are the primary causes of foliage and crop loss eventually affecting the overall economic outcome and yield quality. Hence, various chemical compounds are employed to eradicate the fungi in agriculture. Methods: Virtual screening and molecular docking strategies provide themselves as great alternatives to find lead compounds. Lead compounds for each fungal infection was docked to target protein sequence and assessed for the strongest interaction. Findings: Various molecules were taken under the study, for being the target ligands to bring about a fungicidal reaction in the plant pathogen system. The screening of molecules was done thoroughly to produce the results. Ligands identified through this study allow us to make plant host fight against the fungal pathogen and prevent the occurrence of the disease. The interactions have been thoroughly studied with various softwares like SPDBV and PyMol and through various online databases like STRING, GenePept, PDB, UniProt, PatchDock, Protein structure prediction server -2 and others for the overall evaluation of the drug molecule designed and to study its overall effects for the overall higher efficacy and to prevent the occurrence of the fungal disease and management of the fungal pathogens in agriculture against various economically valuable plants. The lead compounds revealed several hydrophobic and polar contacts were demonstrated by comparing interactions. Applications: The molecular affinity of the fungicidal compound has been tested against the target pathogen as well as the host system components to understand the interaction and to draw out the functioning and the analysis. The compatibility between the molecule and the protein has been studied to decipher the effectiveness of the molecule and its effects in the system. The present results let us establish lead compounds that can be used for the development of antifungal drugs although structural activity relationship studies have to be undertaken.

Transforming growth factor beta receptor 2 single-nucleotide polymorphism association with oral cancer and In silico identification of small drug-like molecules as inhibitors to transforming growth factor Beta-2 receptor

Objective: Oral cancer, in India, constitutes 26% of global oral cancer burden. The major risk factors include tobacco, areca nut, alcohol, and human papillomavirus 16/18; however, only 5%–10% of the high-risk individuals develop oral cancer, indicating the role of genomic variants in susceptibility to oral cancer. Conventional treatment options in oral cancer have resulted in relatively poor prognosis and an unmet need of treatment. In silico analysis, therefore, was performed to identify small drug-like molecules as potential inhibitors of transforming growth factor beta-2 receptor (TGFβRII). Materials and Methods: Seven single-nucleotide polymorphisms (SNPs) were analyzed in 500 histopathologically confirmed oral cancer samples and 500 long-term tobacco users (LTTUs) as controls using allelic discrimination real-time polymerase chain reaction or high-resolution melting analysis. The differential frequencies in oral cancer and LTTUs were calculated using SPSS software (version 19), and odds ratio (OR) to indicate risk to oral cancer using Hutchon.net. structure-based virtual screening of drug-like molecules was performed to identify lead inhibitor molecules to TGFβRII using Schrodinger Suite 2015-4. Results: Heterozygous GC genotype of TGFBR2 rs9843143 demonstrated increased risk ([P = 0.011; OR 1.61 [1.25–2.1]) while CC genotype showed decreased risk (P = 0.005; OR 0.61 [0.44–0.83]) to oral cancer. Increased/decreased risk to oral cancer was not observed for the other SNPs. In silico analysis identified six molecules as inhibitors of TGFβRII kinase domain from 17,723 conformers from Maybridge HitFinder library and 2685 conformers from MEGx AnalytiCon natural product library. Conclusion: SNP rs9843143 (TGFBR2) demonstrated a significant association (P

Cheminformatics-based enumeration and analysis of large libraries of macrolide scaffolds

We report on the development of a cheminformatics enumeration technology and the analysis of a resulting large dataset of virtual macrolide scaffolds. Although macrolides have been shown to have valuable biological properties, there is no ready-to-screen virtual library of diverse macrolides in the public domain. Conducting molecular modeling (especially virtual screening) of these complex molecules is highly relevant as the organic synthesis of these compounds, when feasible, typically requires many synthetic steps, and thus dramatically slows the discovery of new bioactive macrolides. Herein, we introduce a cheminformatics approach and associated software that allows for designing and generating libraries of virtual macrocycle/macrolide scaffolds with user-defined constitutional and structural constraints (e.g., types and numbers of structural motifs to be included in the macrocycle, ring size, maximum number of compounds generated). To study the chemical diversity of such generated molecules, we enumerated V1M (Virtual 1 million Macrolide scaffolds) library, each containing twelve common structural motifs. For each macrolide scaffold, we calculated several key properties, such as molecular weight, hydrogen bond donors/acceptors, topological polar surface area. In this study, we discuss (1) the initial concept and current features of our PKS (polyketides) Enumerator software, (2) the chemical diversity and distribution of structural motifs in V1M library, and (3) the unique opportunities for future virtual screening of such enumerated ensembles of macrolides. Importantly, V1M is provided in the Supplementary Material of this paper allowing other researchers to conduct any type of molecular modeling and virtual screening studies. Therefore, this technology for enumerating extremely large libraries of macrolide scaffolds could hold a unique potential in the field of computational chemistry and drug discovery for rational designing of new antibiotics and anti-cancer agents.

The zebrafish model in oncology

Although cell culture and mouse models will remain a cornerstone of cancer research, the unique capabilities of the zebrafish outline the potential of this model for shedding light on cancer biology in vivo. Zebrafish develops cancers spontaneously, after chemical mutagenesis or through genetic manipulations. Furthermore, zebrafish cancers are similar to human tumors at the histological and molecular levels allowing the study of tumor initiation, progression and heterogeneity. Xenotransplantation of human cancer cells in embryos or adult zebrafish presents the advantage of following cancer cell behavior in vivo. Finally, zebrafish embryos are used in molecule screens and contribute to the identification of novel anti-cancer therapeutic strategies. Here, we review different involvements of the zebrafish model in cancer research.

Screening of potential lead molecules against prioritised targets of multi-drug-resistant-Acinetobacter baumannii – insights from molecular docking, molecular dynamic simulations and in vitro assays

Acinetobacter baumannii, an opportunistic pathogen, has become multi-drug resistant (MDR) to major classes of antibacterial and poses grave threat to public health. The current study focused to screen novel phytotherapeutics against prioritised targets of Acinetobacter baumannii by computational investigation. Fourteen potential drug targets were screened based on their functional role in various biosynthetic pathways and the 3D structures of 9 targets were retrieved from Protein Data Bank and others were computationally predicted. By extensive literature survey, 104 molecules from 48 herbal sources were screened and subjected to virtual screening. Ten clinical isolates of A. baumannii were tested for antibiotic susceptibility towards clinafloxacin, imipenem and polymyxin-E. Computational screening suggested that Ajmalicine ((19α)-16, 17-didehydro-19-methyloxayohimban-16-carboxylic acid methyl ester from Rauwolfia serpentina), Strictamin (Akuammilan-17-oic acid methyl ester from Alstonia scholaris) and Limonin (7, 16-dioxo-7, 16-dideoxylimondiol from Citrus sps) exhibited promising binding towards multiple drug targets of A. baumannii in comparison with the binding between standard drugs and their targets. Limonin displayed promising binding potential (binding energy −9.8 kcal/mol) towards diaminopimelate epimerase (DapF) and UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA). Ajmalicine and Strictamin demonstrated good binding potential (−9.5, −8.5 kcal/mol, respectively) towards MurA and shikimate dehydrogenase (−7.8 kcal/mol). Molecular dynamic simulations further validated the docking results. In vitro assay suggested that the tested isolates exhibited resistance to clinafloxacin, imipenem and polymyxin-E and the herbal preparations (crude extract) demonstrated a significant antibacterial potential (p ≤ .05). The study suggests that the aforementioned lead candidates and targets can be used for structure-based drug screening towards MDR A. baumannii.

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved.

Abstract A34: The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies

Abstract Introduction: It was shown that BCR inhibitors such as ibrutinib interrupt microenvironmental interactions and mobilize malignant B cells of chronic lymphocytic leukemia (CLL) from immune niches into the blood stream. It has been suggested that this mobilization will sensitize malignant B cells to other drugs, providing a possibility to introduce synthetically lethal combinations of drugs with an acceptable toxicity profile. This might be limited to a subgroup of CLL patients with specific underlying molecular mechanisms of disease biology or reaction to BCR inhibitors. The aim of this study was to test for the effect of ibrutinib on the expression of genes and activity of pathways that could be potentially targeted by clinically available drugs to achieve highly potent combinations. Results: We performed gene expression profiling in samples obtained from CLL patients treated with ibrutinib as a single agent (pre-ibrutinib vs. day 15 and/or week 5/12/15; N=12 pairs) to identify potential therapeutic targets. We have primarily focused on analyzes of cell-surface proteins and anti-apoptotic Bcl2 family members, since therapeutic antibodies and small molecular inhibitors for these molecules are available. We have analyzed changes in >30 cell-surface molecules, 8 members of the Bcl2 family, and members of other potentially relevant pathways (chemokines, adhesion, Akt, Erk, Ras, NFkB). Notably, we have observed that the mRNA and B cell-surface levels of CD20 are strongly down-modulated with ibrutinib, which indicates that the previously suggested combination of ibrutinib and anti-CD20 antibodies (such as rituximab or ofatumumab) is likely not ideal. Indeed, we showed that CLL cells with lower levels of CD20 are more resistant to rituximab in vitro, but also in vivo (P<0.01). We also showed that the effect of ibrutinib on CD20 levels is mediated by inhibition of CXCR4/SDF-1 axis, which impairs the SDF-1α (ligand for CXCR4) mediated regulation of CD20 expression (P<0.01). Notably, it has been shown that addition of rituximab to ibrutinib in vivo is able to partially eliminate CLL cells despite lower levels of CD20 suggesting that there must be other mechanism of ibrutinib action that allows for the effect of rituximab. The screening of anti-apoptotic molecules revealed that Mcl1 levels were significantly down-modulated after ibrutinib treatment in vivo (P=0.002), and ibrutinib also inhibited Mcl1 induction in CLL cells co-cultured with stromal cells (HS-5). As Mcl1 was described to directly protect CLL cells from rituximab, we suggest that ibrutinib-mediated Mcl1 reduction is a mechanism that facilitates rituximab efficacy. On the other hand we have shown that several members of Bcl2 family are up-regulated in CLL samples obtained after >3 weeks on ibrutinib therapy. We have also shown that interactions of stromal cells (HS-5) with CLL cells lead to up-regulation of these molecules while ibrutinib inhibits this in vitro (P<0.01). This suggest that the observed in vivo up-regulation of specific Bcl2 family members is due to an adaptation of B cells or selection of malignant B cells with high-level expression of anti-apoptotic molecules sufficient to survive the BCR signalling inhibition. We observed that the survival of CLL cells treated with ibrutinib is dependent on Bcl2, and subsequent Bcl2 inhibition can act synergistically with ibrutinib in B cell lines and CLL samples. These anti-apoptotic molecules therefore represent primary candidates with synthetically lethal potential in combination with ibrutinib. Additionally, we have identified dependence of a subtype of CLL cases on integrin signalling. The inhibition of integrin VLA-4 with anti-VLA4 antibody or integrin-associated kinase overcame the resistance of CLL cells to chemotherapy drugs and also to anti-CD20 antibodies, and synergized with the effects of ibrutinib. Conclusion: We have performed gene expression analysis of primary samples obtained from patients on ibrutinib, and identified gene expression changes that are associated with adaptation of B cells and/or selection of B cell resistant to BCR inhibition. We have shown that malignant B cells are dependent on integrin-signalling and the up-regulation of Bcl2 protein, which makes them synergistically lethal targets. Supported by: The results of this research have been acquired within CEITEC 2020 (LQ1601) project with financial contribution made by the Ministry of Education, Youths and Sports of the Czech Republic within special support paid from the National Programme for Sustainability II funds. Citation Format: Gabriela Pavlasova, Katerina Musilova, Vaclav Seda, Katerina Cerna, Eva Vojackova, Veronika Svobodova, Marek Mraz. The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A34.

Abstract B30: Novel targets for combination therapy in EGFR mutated NSCLC

Abstract Non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR) gene generally show good responses to therapy with EGFR tyrosine kinase inhibitors (EGFR-TKIs). Unfortunately resistance emerges during treatment, driven by different mechanisms. Mutation-driven mechanisms are becoming well understood and include secondary mutations in the EGFR (T790M for 1st generation EGFR-TKIs and C797S for 3rd generation EGFR-TKIs) and activating mutations in NRAS. On the other hand, non-mutation based resistance mechanisms are less well understood but may involve activation of alternative, potentially targetable signalling pathways. To identify alternative signalling pathways and potential combination targets, we performed a bioactive small molecule (n=1902) screen to identify targets that could delay the emergence of resistance to sustained inhibition of EGFR in EGFR-mutant NSCLC cells. 9 out of 186 target classes were significantly enriched (p< 0.05) within the top 2.5 % of screen compounds, when ranked for their ability to sensitise cells to the 3rd generation EGFR-TKI AZD9291. We identified expected hits such as AKT, PI3K and mTOR but also novel targets. To prioritise targets for in vivo validation, compounds were tested in 3D-spheroids, which have a complex microenvironment, providing a more tumour-like model. Most compounds were more cytotoxic in 3D compared to 2D when used individually, preventing or slowing down spheroid growth. EGFR-TKI combination effects in 3D varied when compared to 2D-models. FAK inhibition was less effective in spheroids compared to 2D. AKT or MEK inhibitors showed marked effects in combination, further delaying spheroid growth under EGFR-TKI treatment but also inhibited spheroid growth when used on their own. Inhibitors of novel targets were identified that showed promising combination effects even at sub-toxic single-agent doses. Our data identify new candidates for combination therapy with EGFR-TKIs and suggest that 3D-spheroids may be a useful approach for prioritising combinations for in vivo studies. Citation Format: Neele Drobnitzky, Agata Patel, Amalina Mumin, Fiona Cahill, Luiza Madia Lourenco, Samantha Olyha, Sureyya Corbacioglu, Yanyan Jiang, Anderson J. Ryan. Novel targets for combination therapy in EGFR mutated NSCLC [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B30.