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High Throughput Screening (HTS) and Affinity Selection-Mass Spectrometry (AS-MS) for Early Drug Discovery

Yingjie Zhu, Drug discovery support – Sr. Director, WuXi App Tec


High throughput screening has been the corner stone of hit identifications for new and existing biological targets. The conventional high throughput screening relies on a large "druggable" compound collection and the availability of screening quality assays. Recent technology advancements give alternative approaches for initial hit identification. DNA encoded library is a technology for synthesis and screening on unprecedented scale of collections of compounds. The compounds are synthesized by combinatorial chemistry and bar coded by short DNA sequences. In principle this technology will allow the isolation of binders to a wide range of proteins historically difficult to tackle due to either lack of screening assay or lack of target suitable compounds in the collection. Hits coming out of the DEL screening will need to be tested for target specificity and biological function. The biological activity conformation on the hits identified through DEL screening has been very challenging. Affinity Selection-Mass Spectrometry is a sensitive technology for identifying small molecules that bind to target proteins. It takes advantages of conventional "druggable“ compound library and pull hundreds of compounds into one mixture. Siince the Mass Spectrometry can detect the hundreds of compounds in one mixture in one run, AS-MS offers higher throughput and lower cost approach than conventional HTS. Like DEL, hit from AS-MS will also need to be confirmed for target specificity and biological functional activity. The conventional HTS screens compounds with either a target specific biochemical assay or a functional cell based assay. The hit identified from the "druggable" library often form structure-activity relationship (SAR) right away to guide the acquisition or synthesis of desirable new compounds for better potency and efficacy. AS-MS is a valuable complement to traditional high throughput screening. Several conventional HTS and AS-MS case studies are presented.



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Directly Linking Single T Cell Phenotype and Function to Genotype on the Lightning Platform

Mark White, PhD – Senior Director, Marketing, T Cell Discovery, Berkeley Lights Inc


T cell therapies for cancer treatment are challenging to develop because of the complex mechanisms and cell interactions that underly T cell-mediated tumor killing. Current technologies rely on correlating phenotype, function, and gene expression based on experiments performed on different populations of T cells because no one platform is able to assess cell surface marker expression, cytokine secretion, and tumor cell killing activity of the same T cell and recover this cell for downstream genomic analysis. Here we share two use cases - CAR-T cell functional screening and TCR sequence recovery following functional assay - that demonstrate how the T Cell Analysis Suite on the Lightning optofluidic platform can be used to directly link T cell phenotype and function (IFNγ secretion and tumor cell killing) to genotype (TCR sequence recovery) at a single-cell level and on the same T cell, enabling deeper and more thorough characterization of how T cells mediate tumor cell death and potentially the development of more efficacious therapies.



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Cell painting for compounds clustering and Mechanism of Action characterization

Pierrick Rochard – Group Leader, Evotec


Cell Painting is a multi-parameter image-based description of the cell response to any perturbator condition: treatment with a compounds, a siRNA, CRISPR engineering. We have implemented Cell Painting workflow at Evotec to allow for the study of several thousands of compounds, using automated process for cell treatment and labelling, image analysis, data processing and quality control. We have validated the technology by studying a collection of 5,200 bioactive compounds and evidenced clustering of compounds sharing similar targets/pathways. We are now applying Cell Painting taking advantage of a reference set of compounds targeting specific pathways within the biological landscape.



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CSox-based Sensors for Continuous, Homogeneous and Quantitative Monitoring of Protein Kinase and Phosphatase Activity

Erik Schaefer, PhD – President, CEO & CSO, AssayQuant Technologies, Inc.


Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of more than 400 diseases. Currently, 30% of all drug development efforts are focused on protein kinases. Although 51 drugs are approved and >200 in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been an expanded focus on kinase inhibitors with different modes of action, where new tools are needed to effectively characterize inhibitor mechanism of action, predict drug potency and to drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of peptide sensors that can be used for the direct, continuous, quantitative and homogenous detection of kinase and phosphatase activity with recombinant enzymes and crude cell or tissue lysates to enable discovery, drug development and precision medicine. Methods: We harnessed chelation-enhanced fluorescence by combining next generation sulfonamido-oxine (Sox) chromophore technology with high-throughput solid-phase peptide synthesis methods to identify optimized physiologically-relevant substrate sequences, where enzyme activity is monitored kinetically using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode using Europium and time-resolved fluorescence (TRF, Ex/Em 360/620 nm). Results: We demonstrate the ability to rapidly identify novel optimized sensor sequences with improved performance including highly-generic substrates for robust detection of 80 tyrosine kinases (EGFR, Her2, BTK, ITK, JAK etc.) and highly-selective substrates for quantitative detection of targeted kinases in crude cell or tissue lysates (ERK1/2 MAPKs, CDKs, PIM). With protein phosphatases, CSox-based phosphopeptide substrates are used to monitor activity with specificity for tyrosine (PTP1B, PTPN2, SHP1/2) or serine/threonine (PP2A, PP2B, PP2C, PHLPP). Conclusions: The generation of robust activity-based sensors, even where peptide assays previously weren’t available, opens new areas for effective drug discovery. The CSox sensor-based Kinetic assay format is ideal for elucidating drug mechanism of action, potency and enzyme regulation. The Europium/Red endpoint TRF format is ideal for HTS, SAR and profiling. Together, these formats can be applied across the entire discovery and drug development workflow using the same optimized sensor substrate sequence, providing a quantum improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors.



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Development of a 3D High-Throughput Robotic Screening Platform

Drew Gunderson – Senior Automation Engineer, GNF


GNF has begun developing systems utilizing collaborative robots to enable a high degree of flexibility in both layout and functionality. The systems are user reconfigurable which is ideal for rapid testing of prototypes and then running these new assay technologies on a high-throughput scale. Here we highlight work done to enable high-throughput dispensing and screening of cells grown in 3D culture. The 3D System utilizes existing GNF Systems technologies such as the 1-Tip-Dispenser (1TD) which has been modified and enclosed in a robot-accessible environmental chamber to achieve non-contact droplet ejection of 3D reagents as well as the new GNF Systems Plate Curing Station for photopolymerization of hydrogels in assay plates. This poster will describe the development strategy and end result of our efforts to enable high-throughput, complex 3D assays on a novel robotic platform.



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In‐house well plate heating system development for SplitLuc CETSA assay, and improving heating andcooling efficacy of a Thermo‐Fisher thermal mixer adapted for flat‐bottom well plates

Eric Wallgren – Prototype Instrumentation Lead, National Institutes of Health - National Center For Advancing Translational Sciences


Achieving the required parameters of accuracy, uniformity, repeatability and speed when heating and/or cooling multi‐well microtiter plates can be difficult if no commercially available instrument exists. Such was the case for Dr. Mark Henderson’s SplitLuc CETSA assay for which a novel heating system capable of RT to 100 C, 1% temperature accuracy, and sufficient thermal mass combined with rapid initial heating and make‐up heating, that when the plate is loaded in the heater, it is brought to temperature in under 1 minute, and is retained in intimate contact by a clamping plate with 4 thumbscrews and a silicone foam elastomeric sheet to assure uniform pressure with the heat source to avoid edge effect. Initial proof of concept of a simple aluminum block on a VWR hot plate worked well enough for initial assay development. The solution is a copper block that has been machined to accommodate a flat bottom 384‐well plate. The block’s heat source is a 120 volt, 110 watt, 5 watt/square in film heater applied to the underside of the machined copper plate nest block. Heater control is effected by a programmable mini controller and a type T thermocouple mechanically fastened inside the copper block near the center of thermal mass. Critical to this placement is the construction of the plate assembly where a recess is machined in the plate facing side of the block with a hole for the thermocouple exiting the end of the block. Once the thermocouple is installed, a close fitting copper plug is pressed in place, peened over, and machined flush with the surface. The thermocouple placement assures temperature stability without spikes or dips. Concurrent with this heater effort, parallel work necessitating both heat and agitation lead to adapting a Thermo‐Fisher thermal mixer with a similar copper block and clamping system.



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Beyond High Content Screening: An Open Next Generation Image Analysis Platform

Nathan Hotaling – Senior Data Scientist, National Center for Advancing Translational Science


There is an increasing interest in discoveries from images acquired by high-throughput and high content microscopy imaging of multi-well plates with biological specimens under a variety of conditions. As multi-dimensional automated imaging increases its throughput to thousands of images per hour, the computational infrastructure for handling the images has become a major bottleneck. The bottleneck associated challenges arise due to big image data, complex phenomena to model, non-trivial computational scalability that leverages advanced hardware and cutting-edge algorithms, and incompatible software tools that vary in the language they were written in, platform they were written for, and capabilities they were designed to execute. To address these interoperability and visual exploration challenges, the National Institute of Standards and Technology (NIST) and the National Institutes of Health (NIH) - National Center for Advancing Translational Science (NCATS) have formed a close collaboration to develop an open source platform for executing web-based image processing pipelines over very large image collections with interoperable plugins. The plugins developed by both institutes are based on software containers as standardized units for server-side deployment, as well as on dynamically created web user interfaces (UI) to enter parameters needed for the software execution and for advanced visual data explorations on the client side. Each container packages code, with all its dependencies, and has an entry point for running the computation in any computing environment. Each UI description file contains metadata about the plugin container and the computation parameters. We demonstrate the utility of the platform with algorithmic plugins by analyzing 20x 1536-well plates with three spectral channels and a single field of view (FOVs) per well to determine the dose response of SOD1 knockout cells to several drug libraries. We analyzed over 92,000 images, created an interface to easily view/interact with the >32 gigapixels of image data simultaneously, and developed workflows that not only enabled this visualization but incorporated state-of-the-art neural networks for segmentation and machine learning algorithms for dimension reduction of extracted features from these segmentations. Workflows consisted of over 50 interoperable, chained, containerized plugins derived from developers at both NIST and NCATS. In addition, fully integrated Jupyter notebooks accelerated prototyping and parameter settings while interactive and scalable scatterplots helped visualize and subset the high dimensional data obtained from the extracted features of the segmentations of the cells. Demanding computations were supported via batch processing and deep learning-based pipelines were executed on GPU enabled hardware. The execution of the analysis was performed in AWS showing the feasibility of using modern horizontally scalable hardware for large image datasets. With NIST’s and NIH-NCATS’ combined efforts, researchers are now enabled to discover quantitative insights from their imaging data and reuse computational tools developed by anyone following the web computational plugin conventions.



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Development of a gold nanoparticle-based colorimetric sensor for a sensitive endotoxin detection in aqueous solutions

Saskia Reichelt, M.Sc. – Research associate, Technische Universitaet Dresden


A recent study estimated: there are more than 250,000 deaths caused by sepsis every year alone in the United States. Lipopolysaccharides, which are located in the outer membrane of gram-negative bacteria, are the major cause. However, not only bacterial infections induce this strong immune response but also endotoxins on implants, within the tubing, syringes or fluids like vaccines or dialysis water. The threshold for developing sepsis, which might lead to multi-organ failure and therefore death, is around 4 ng endotoxin per kg bodyweight. FDA-approved test systems are based on animal components (blood of the horseshoe crab), or even animal experiments (rabbit pyrogen test) are performed. Another disadvantage is that the experiments can take hours up to several days. Consequently, there is an urgent necessity for a rapid bedside test, even if it can only give an initial indication of contaminated products or infected patients.
At the SmartLab system group of the TU Dresden, Germany, we have developed a colorimetric sensor based on self-synthesized gold nanoparticles. The increase of agglomeration of 15 nm gold nanoparticles at a given salt concentration correlates with decreasing amount of endotoxin in the sample.
We currently have achieved a detection limit of 1 EU/ml, which corresponds to 0.1 ng/µl in an aqueous solution. The entire test takes approximately 15 minutes, whereas the preparation of the liquid sample needs an additional 15 minutes. The specificity of the test system is addressed by the utilization of aptamers. These short artificial nucleic acid sequences (25 – 90 bases, around 25 kDa) are identified by SELEX. This selection process is performed by exposing the target structure to an aptamer library followed by harsh selection pressure (e.g. washing steps).
Samples with endotoxin levels expected to be below 1 EU/ml and since blood samples need to be diluted due to salt concentrations, we aimed for an upstream concentration step. In cooperation with an industrial partner, we engineered a metalized high-performance membrane. The electrical potential of the membrane can be actively controlled while the membrane itself is fitted into a modified syringe filter holder. The negatively charged endotoxins adsorb on the functionalized membrane. After re-dissolving the molecules are available for the gold nanoparticle-based rapid test. A sample volume of 20 µl is sufficient for single testing. Once the assay components have been mixed, the result can be interpreted visually within two minutes at the point of care without any further instruments.



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A novel, robust, label free system for measuring post translational modification of proteins.

W. Adam Hill, Dr – Partner, Triple Sharp Venture Engineering


While your genome defines possibilities and your transcriptome defines what’s likely, it is your proteome that defines your destiny.  To date, most measurements of proteins have focused on capturing a single specific epitope within a protein and measuring abundance.  While this, in some cases, has proven effective in diagnostics, the variations and “fingerprints” of post translational modification have often been lost by the binary nature of the readout.

This presentation will demonstrate, using three exemplar data sets, the power of multiplexed affinity capture mass spectrometry to look beyond the abundance of the captured epitope and evaluate other changes in the proteins and pathways of interest. 

Pathway profiling data from a variety of kinase inhibitors clearly show differential changes in abundance and phosphorylation state, not only of the purported target of the inhibitor but also downstream proteins.  This data can be used to guide medicinal chemistry towards the most efficacious molecular profile, rather than a monotheistic measurement of potency at one target.

Profiling of digested brain homogenates can distinguish diseased from normal brains – elucidating potential mechanisms for protecting against neurodegeneration.  The quantitative data presented clearly demonstrates multiple modifications that occur in the disease state and the relative ratios of post translational modifications are significantly altered.

The third example demonstrates the ability of affinity selection mass spectrometry to identify covalent binders to a protein of interest.  This highly sensitive approach (fmoles of protein) enables tracking of PK /PD for a covalent therapeutic, as well as percent occupancy.

            Affinity capture mass spectrometry using spatially defined arrays is a powerful, high throughput technique that has application throughout the drug discovery process.  It’s high sensitivity and fidelity along with its ability to identify post translational modification without an antibody to the specific modification is an enabling technology in the quest for new and better medicines and diagnostics.



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Environmentally friendly manufacturing of flexible graphite electrodes for wearable zinc monitoring in sweat.

Cyro Lucas Chagas, Post-doctoral fellow – Post-doctoral Fellow, Universidade de Sao Paulo


Electrochemical sensors based on graphite and polymers have emerged as powerful analytical tools for bioanalytical applications. However, most of the fabrication processes are not environmentally friendly since they often involve the use of toxic reagents and generate waste. This study describes an alternative method to produce flexible electrodes in plastic substrates using graphite powder and thermal laminating sheets by solid-solid deposition through hot compression, without the use of hazardous chemical reagents. The electrodes developed through the proposed approach have successfully demonstrated flexibility, robustness, reproducibility (RSD around 6%) and versatility. The electrodes were thoroughly characterized by cyclic voltammetry, electrochemical impedance spectroscopy, Raman spectroscopy, and scanning electron microscopy. As a proof-of-concept, the electrode surfaces were modified with bismuth and used for zinc analysis in sweat. The modified electrodes presented linearity (R² = 0.996) for a wide zinc concentration range (50-2000 ppb) and low detection limit (4.31 ppb). The proposed electrodes were tested using real sweat samples and the achieved zinc concentrations did not differ statistically from the data obtained by atomic absorption spectroscopy. To allow wearable applications, a 3D printed device was fabricated, integrated with the proposed electrochemical system and fixed at the abdomen through elastic tape to collect, storage and analyze the sweat sample. Matrix effect test was performed, spiking the real sample with different zinc levels, and the recovery values varied between 85 and 106%, thus demonstrating adequate accuracy and robustness of the flexible electrodes developed based on the proposed fabrication method.



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Novel MALDI-TOF MS Workflow for Ultrahigh-Throughput Screening of Different Analytes at Each Position on a Plate

Sergei Dikler, PhD – Technical Project Manager, Bruker Corporation


MALDI-TOF mass spectrometry was established as a label-free ultrahigh-throughput screening approach for biochemical assays where substrates and products were small molecules, peptides and proteins. In this case the same list of analytes was monitored at every position on a MALDI plate. More recently, we extended this approach to screening of synthetic chemical reactions, which required monitoring of different small molecule starting materials and products at every position on a MALDI plate. This necessitated the development of a novel software workflow and optimization of data processing methods for a dramatically increased number of analytes. This work focuses on application of the new Synthesis Screening workflow not only to screening of chemical reactions but also to screening of a custom library of small molecule pharmaceuticals.

A custom library of 41 small molecule compounds was analyzed in automated mode on a MALDI-TOF/TOF system equipped with a 10 kHz scanning beam laser. The automated runs were set up, monitored and visualized in MALDI PharmaPulse 2.2 software that included the novel Synthesis Screening module. The new software workflow allowed monitoring of up to 100 different analytes per position on a MALDI plate prepared in 384, 1536 or higher density formats. 

Runs of a MALDI plate prepared with the custom library compounds in 384 format resulted in 0.67 failed plate positions per run (averaged from 3 runs). Five compounds from the custom library were monitored as sodium adducts [M+Na]+ while the remaining compounds were detected as [M+H]+ ions. The software allowed monitoring of adduct ions selectively for specified analytes of interest or for all analytes. The workflow with detection of multiple different analytes at each position on a MALDI plate is applicable not only to synthetic chemical reactions but also to checking compound libraries and multiplexed biochemical assays.



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Metabolomics: A Strategy for Monitoring Partial Enteral Nutrition Treatment and Identifying Metabolic Signatures of Inflammation in Paediatric Crohn’s Disease Patients in Remission

Engy Shokry, PhD – Postdoctoral researcher, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children's Hospital , LMU - Ludwig-Maximilians University of Munich


Metabolomics: A Strategy for Monitoring Partial Enteral Nutrition Treatment and Identifying Metabolic Signatures of Inflammation in Paediatric Crohn’s Disease Patients in Remission

Engy Shokry, Ph.D.
Ludwig Maximilians University of Munich
Munich, Germany

Co-Authors
Jair Marques, Annecarin Brueckner, Tobias Schwerd, Sibylle Koletzko, Berthold Koletzko

Growing evidence suggests that partial enteral nutrition (PEN) is successful in maintenance of remission in Crohn’s disease (CD) patients, however the mechanism remains unclear. Concomitantly, it is believed that metabolic changes underlie the disease-related inflammation status. We aimed to identify some metabolic relationships relevant to CD and how they respond to PEN therapy. Within a non-randomized controlled intervention study, plasma samples were obtained from paediatric CD patients in remission quarterly over one year. The intervention group received a casein-based formula, providing ~25% of daily calorie intake. Targeted metabolomics was applied to measure >400 metabolites comprising phospholipids (PL), non-esterified fatty acids (NEFA), acyl carnitines (AC), Krebs cycle metabolites, amino- and organic acids. For the statistical analysis, a multilevel two-factor sparse partial least squares (spls) was applied for group discrimination and identifying sources of variation. Another aspect of the analysis was to investigate the metabolites most correlated with inflammation markers (C-reactive protein (CRP), erythrocyte sedimentation rate, leucocyte count, calprotectin and resistin). Accordingly, an integrative spls was performed using the matched data sets. Non-PEN/PEN groups were completely separated at all time points and phosphatidylcholines (PC), especially those comprising palmitic acid (PA) were identified as biomarkers for consumption of the formula where PA is the major fatty acid. Differences in PC were highest at 3 months of the intervention pointing out to initial higher patients’ compliance during the first quarter of the study when patients are most motivated. Interestingly, consistently lower levels of acyl-lysophosphatidylcholines diacyl-phosphatidylcholines ratio (Σlyso.PCa/ΣPCaa), a recognized metabolic biomarker of inflammation, were found in PEN relative to Non-PEN indicating its potential contribution to the resolution of inflammation observed with PEN therapy. Apart from the intervention, metabolic changes involving phospholipids comprising polyunsaturated fatty acids (PL-PUFA) and lactic acid were detected in both PEN/Non-PEN patients during the course of the study which may be related to the disease progress. They increased remarkably at the end of the study, despite being stable in both groups throughout the study duration. Several NEFA and mid-/short chain AC and fatty acid β-oxidation markers were found to be highly correlated with all investigated inflammation markers, principally CRP confirming the contribution of lipid metabolism in the underlying inflammatory processes in CD. In conclusion, our study was capable of identifying biomarkers for continuously monitoring patient’s compliance with PEN treatment, thus overcoming inaccurate patient self-reporting. It also detected temporal metabolic changes in patients through remission which could help in detailed assessment of disease progress and thus prediction of relapse.



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A liquid chromatography high-resolution mass spectrometry in vitro assay to assess metabolism at the injection site of subcutaneously administered therapeutic peptides.

Alberto Bresciani, PhD, n/a – IRBM


In this work the set-up, validation and application of a new LC-HRMS-based in vitro assay to assess peptide metabolism at the subcutaneous injection (SCiMetPep assay) is presented. Subcutaneous injection (SC) is the most common administration route for peptide therapeutics and, in some cases, catabolism at the injection site can significantly affect ADME properties, especially bioavailability. The SCiMetPep assay was developed using human, rat and Göttingen minipig SC tissue homogenate supernatant, and allows for both determination of degradation rate (half-life) of the parent peptide and identification of metabolites generated from enzymatic proteolysis. The SCiMetPep assay was validated using known peptides, like GLP-1 analogues and a new series of structurally related peptides and a fairly good correlation was found between SC metabolic stability and bioavailability.

This assay represents a valuable preclinical in vitro tool to predict the ADME properties of peptide therapeutics at an early stage of drug discovery.



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Protein liposome binding using the FIDA technology: A new approach for membrane protein interactions

Morten Pedersen – Sr. application scientist / Industrial PhD Fellow, Fida Biosystems


Liposomes are artificial vesicles composed of lipids. Liposomes has found widespread applications within drug delivery and as models for cell membranes. The size, charge and membrane dynamics of liposomes can be controlled in detail during the preparation process and they are therefore an ideal platform for systematic studies on protein-membrane interactions. The size range (hydrodynamic radius) of liposomes ranges from 20 nm to more than 200 nm, which makes them challenging to study using a single analytical methodology.

Flow Induced Dispersion Analysis (FIDA) is a new size based analytical platform which enables size and binding (Kd) to be assessed in a single technology. FIDA is based on Taylor dispersion in thin capillaries which enables molecular and particle sizes, in the range of 0,2nm – 500 nm, to be assessed.1-3 This size range covers small molecules, peptides, proteins and larger particles such as liposomes and extracellular vesicles. Using a fluorescent label on a ligand interacting with a protein or particle, FIDA measures in-solution degree of binding which can be translated into affinity constants (Kd´s), ligand and complex size. FIDA measures in complex sample matrices such as plasma, serum and fermentation broth and is fully automated.

In this work we have studied how the protein a-synuclein interacts with membranes by using liposomes as a model system. The understanding of a-synuclein –membrane interaction is highly relevant for the understanding of a-synuclein aggregate formation and therefore for understanding the progression of Parkinson’s disease. It was possible to quantify the interaction as well as to measure the in-solution size of the a-synuclein – liposome complex. Further, competition experiments using a-synuclein fragments provided insights into the location of the interacting residues of a-synuclein.

The methodology develop in the present work may easily be extended to membrane proteins as well as to other vesicles such as for example exosomes and virus particles.

References

[1] Poulsen et al, Analyst, 2015, 140, 4365.

[3] Poulsen et al, Analytical Chemistry, 2016, 88, 9056.

[3] Pedersen et al,  Analytical Chemistry, 2019, 91, 4975.



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Application of a novel in vitro drug distribution profiling assay in drug discovery projects.

Fredrik Wagberg – Associate Principal Scientist, AstraZeneca


To understand the mechanism of a drug molecule and to translate the efficacy of the drug between in vitro and in vivo models require knowledge of a numbers of properties of both the compound and its biomolecular target. In drug discovery an incomplete understanding of these properties is a common reason for lack of efficacy when a drug is tested in clinical settings. In order to be efficacious a drug need to be present at sufficient concentration at the physiologically relevant site of action, i.e. where its target is located. Furthermore, only the unbound drug is available for interaction and hence, this is a valuable parameter to understand. While there is reasonable consensus that, at steady state, unbound compound concentration inside cells in in vivo settings can be assumed to equal the unbound plasma concentration, this correlation is still not very helpful when trying to understand data from cellular in vitro models, especially when the biological events are faster than the time needed to establish equilibrium. Measuring the unbound concentration of compound inside a cell poses several experimental and analytical challenges. Specifically, to extract a relevant cellular cytosol without dramatic perturbation to the cells or established equilibria is extremely challenging. Also, detecting low intracellular concentrations is analytically challenging from small cell numbers, often limiting the use of disease-relevant cells at enough capacity to support drug discovery programs. To this end we have built a LCMSMS method that measures how compounds distribute within cellular and extracellular compartments.  The protocol was optimized to minimize perturbation to cells while extracting the cell cytoplasm. To validate the method, we assessed cellular compartment leakage from permeabilized cells using Western blots towards representative nuclear, membrane-associated, mitochondrial and cytoplasmic proteins. A combination of short wash procedures (< 1min) and acoustic dispensing was used to minimize assay times, and thus impact on equilibria, and to prevent non-specific binding associated with contact dispensing. Furthermore, all data was normalized to compound standards to enable absolute quantitation. To our knowledge this is the first method that gives a quantitative profile of how compounds distribute across different compartments of cell and with 384-well capability to support drug discovery efforts at scale. While the original question, i.e. “what is the unbound intracellular compound concentration”, remains unanswered we believe this cellular distribution profile provide valuable insight to the interpretation of in vitro cellular data and potentially translation to in vivo models. This contribution puts the cellular distribution profile in perspective of critical physicochemical compound properties and exemplifies its application to select drug discovery projects.



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Highly Sensitive ELISA's in 90-minutes

Catherine Wark, PhD – Applications Manager, BMG LABTECH Ltd.


Since its introduction in the early 1970’s the enzyme-linked immunosorbent assay (ELISA) has become one of the most reliable and relied upon biochemical assays. Detecting the presence (and quantity) of an analyte has proved an invaluable tool as a diagnostic in medicine or in basic research spanning nearly every biological discipline.
The main drawbacks of the ELISA approach have always been the relatively low throughput and the tedium of multiple wash steps that normally take 3-5 h to perform. Abcam’s SimpleStep ELISA® technology addresses these issues by streamlining the process to a semi-homogeneous format. The key change is the use of an anti-tag immobilization antibody and the end result is a simple 90-minute, single wash protocol. Here, we describe the detection of this innovative ELISA by the SPECTROstar Nano. When combined with MARS data analysis you will get your great results in the fastest, easiest way possible. 
Examplary data are shown for three important targets:Human Pro-Collagen I alpha, Human Interleukin 6 (IL6), and Green fluorescent protein (GFP). All were detected with excellent sensitivity in one third of the time it would take to complete a typical ELISA.



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Panning for Metals

Jarrod Walsh – Associate Principal Scientist, AstraZeneca


There are many common misconceptions concerning High Throughput Screening (HTS). One such is that finding actives is hard. In reality this is usually the easy part. The real challenge lies in sifting out those gems the chemist should be focused on from the wasteful dead ends. Many sources of undesirable hits exist whether they be technology artefacts, redox cycling compounds, inhibitors of coupled enzyme systems or any other from a myriad of mechanisms. Based on our screening experiences in AstraZeneca one of the most common and difficult to identify sources is contamination of compound samples. Chief amongst those is metal contamination retained from the compound synthesis. In this presentation we describe the positive impact the Acoustic Mist Ionisation – MS (AMI-MS) technology is having on minimising our false positive detection technology artefacts. Particular focus is paid to the creation of a novel scavenger-based AMI-MS assay that enables detection and characterisation of these elemental contaminants. In parallel to this work we discuss the creation of a bespoke annotated compound set utilised to profile all targets and assay systems entering the HTS portfolio. This sample deck has proven invaluable in identifying a target’s specific susceptibilities. We explore how our different project outputs have been enriched by these impurities, what it’s shown us about our HTS collection’s metal liability and strategies to deal with it.



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A Phenotypic Screen for Compounds that block cAMP-mediated Immunosuppression in the Tumor Microenvironment.

Adam Zweifach, PhD – Associate Professor, Dept. Molecular and Cell Biology


Overcoming tumor suppression of immune responses is vital for treating cancer. Three major tumor immune-suppressing factors - PGE2, adenosine and tumor associated Tregs – increase intracellular cAMP in effector T (Teff) and natural killer (NK) cells to inhibit their tumoricidal activity. PGE2 and adenosine activate EP2/ EP4 and adenosine A2 receptors, respectively, stimulating adenylyl cyclase (AC) in effector cells. Tregs transfer cAMP directly into the cytoplasm of effector cells via gap junctions. cAMP activates cAMP-dependent protein kinase (PKA), which phosphorylates and activates csk, a tyrosine kinase with homology to src-dependent protein kinases. Csk in turn phosphorylates and inactivates src-family tyrosine kinases required for effector cell activation. None of these components are particularly attractive drug targets, but phenotypic screening offers the possibility of identifying additional currently-unknown targets for therapeutic intervention.

 

We previously used TALL-104 human cytotoxic T cells and high throughput flow cytometry (HTFC) to conduct no-wash assays for immunosuppressants and for compounds that enhance T cell receptor-stimulated responses. TALL-104 cells express EP2 and EP4 receptors. Treating them with PGE2 increases intracellular cAMP. Adding IBMX, an inhibitor of phosphodiesterases that degrade cAMP, further increases cAMP. Manipulations that increase cAMP (PGE2 +/- IBMX, forskolin, or membrane permeant cAMP analogs) all inhibit granule exocytosis. The magnitude of effects of different treatments on cAMP and inhibition of granule exocytosis are correlated, with the combination of PGE2 and IBMX being most effective. Treating cells with PGE2 + IBMX potently suppresses target cell killing.

 

We exploited these features to create an assay for compounds that inhibit cAMP-mediated immunosuppression. We treat TALL-104 cells with PGE2 and IBMX, then add anti-CD3 beads to trigger lytic granule exocytosis. Anti-LAMP-1 antibody in the extracellular solution is used to detect lytic granule exocytosis without washing via HTFC. Cells bound to stimulatory beads are identified by gating on scatter. The percentage of responding cells is determined using unstimulated controls to set analysis gates. In 96-well plates laid out with alternating columns of DMSO and PGE2/IBMX treated cells, Z’ is routinely > 0.5. We have transferred the assay to the University of New Mexico Center for Molecular Discovery for pilot screening in 384-well plate format using an automated work flow and true hypercyt-enabled HTFC. Z’ was  > 0.5 in test plates of controls and in preliminary screening of the Prestwick compound library.

 

We intend to screen a library of diverse lead-like molecules, confirm activity of hits on granule exocytosis using an orthogonal assay and then test effects on target cell killing. For confirmed hits, we will not pursue those that prevent PGE2-stimulated generation of cAMP, prioritizing instead those that block suppression of responses when cAMP is increased, as these will work by unknown mechanisms and target three important immune-suppressing factors.



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Breaking the Tyranny of Z’: Analyzing the significance of a ubiquitous assay performance metric.

The assay quality metric Z’, defined as 1-(3*(σpc+ σnc))/((μpcnc)) where σ and μ represent standard deviation and  mean, respectively, defines a separation band between normalized positive (pc) and negative control (nc) populations (Zhang et al J. Biomol. Screen. 1999). Z’ has come to determine whether assays are considered suitable for high throughput screening (HTS)- it is now almost an absolute requirement that Z’ be > 0.5 for an assay to go forward. However, this seems to result only from series of qualitative descriptions used in the original publication, not an analysis of assay performance. Critically, requiring Z’ > 0.5 has negative consequences for HTS. It almost certainly disqualifies valuable cell-based and phenotypic assays, as these are often inherently more variable than biochemical assays, and it may lead researchers to conduct assays under unfavorable conditions that prevent them from finding compounds.

 

We analyzed the effects of Z’ on assay performance. Assuming equal sample variance, we can set the Type I error probability α (probability of false positives) to various levels and calculate the minimum inhibition that can be detected at a given power, 1-β, for assays with Z’ ranging from 0.1- 0.9. Assays with Z’ < 0.5 behave surprisingly well. For example, setting α < 0.05, an assay with Z’= 0.4 can detect inhibition levels ≥ 24 % with power of 0.8, while an assay with Z’ of 0.1 detect inhibition levels ≥ 36%. Higher activity levels are required for higher power or lower α. With α < 0.001 (corresponding to > 3σ) and β = 0.8, an assay with Z’ = 0.1 can still detect inhibition levels ≥ 58%.

 

These results indicate that assays with Z’ < 0.5 can likely be used to identify compounds of interest, provided activity cutoffs appropriate to assay performance are chosen. We have devised a mixture model for matching assay performance to any desired level of α and power, controlling the false discovery rate. The only assumptions are that (i) uninhibited controls or inactive compounds have a normal distribution with mean 1 and variance σ2 and (ii) a negligible number of active compounds have activity ≥ 1. We provide freely available software in R that includes clear and concise graphical tools that show the trade-off between the power and the false positive rate for any Z’. We suggest that rather than simply require Z’ > 0.5, tools like those we provide be used to define for a given assay the activity cutoff that will be applied and the expected number of false positives. Doing so will allow phenotypic and cell-based assays that are currently disadvantaged by current practice to go forward in an informed way and may ultimately help reveal new leads for important diseases.



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A standardized and scalable angiogenesis assay using iPSC-derived endothelial cells. Enabling phenotypic screening in cardiovascular drug discovery

Arie Reijerkerk, n/a – Ncardia


It is well recognized that angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays a fundamental role in health and disease. For the discovery of new drugs targeting the vasculature, it is common practice to use primary endothelial cells in culture systems but, a number of constraints impede their routine application in drug discovery. This includes the limited supply and batch-to-batch variations due to genetic and other variabilities amongst donors. This introduces an inherent biological variability component that is difficult to control and which may negatively impact assay reproducibility.

To meet the demands of predictive preclinical vascular drug research, improved in vitro models of the vasculature are required: assays that are amenable to high-throughput screening, with a scalable and robust cell source, in a physiological relevant cellular micro-environment. Human induced pluripotent stem cell (hiPSC)-derived cells of the cardiovascular system are promising candidates to fulfil these requirements.

We have developed a reproducible manufacturing process for the generation of large numbers of iPSC-ECs ( >90% CD31+) using a set of controlled bioreactor systems and subjected the cells to a standardized microfluidic cell culture platform (OrganoPlate) to develop an angiogenesis assay which is compatible with high-content imaging and high-throughput screening.

Upon the formation of vascular lumen in microfluidic channels, sprouting into a three dimensional collagen-based matrix was triggered with an optimized gradient of angiogenic factors. Total sprout  area, total sprout length and migration distance of each sprout were quantified using high content imaging. We demonstrate that, similar to primary endothelial cells, iPSC-ECs reproduce important hallmarks of angiogenic sprouting, including the formation of tip cells that display their characteristic filopodia and trailing stalk cells that finally anastomose and form a perfusable lumen. Complete anastomosis coincided with a significant decrease in permeability for a fluorescent tracer, indicating maturation of the vessel structures. At the same time non-mature angiogenic sprouts retracted and pruned.

Remarkably we detected known anti-angiogenic compounds such as Sunitinib (a clinically used VEGFR2 inhibitor) at an IC50 of 21 nM which is exactly in line with observations in a target based assay. The transient glycolysis inhibitor 3PO, also known for its anti-angiogenic activity similarly reduced the sprouting of hiPSC-ECs in a concentration-dependent manner, suggesting that the process is VEGF-driven with glycolysis as main energy source. The developed assay is robust (AW > 10) and reproducible (Z-factor > 0.7), providing excellent opportunities for predictive phenotypic compound screenings in drug discovery.

Altogether, the combination of a standardized microfluidic cell culture platform with a scalable and robust cell source is a major step in the standardization of physiologically relevant in vitro angiogenesis assays, as it offers the required robustness, compatibility and scalability to be integrated within drug screening.



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