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Cancer therapy is increasingly becoming individualized, but there are also big gaps between the molecular knowledge of individual cancers we can generate today and what can be applied in the clinic. In an attempt to bridge this knowledge gap between cancer genetic and molecular profiling and clinically useful information, an individualized systems medicine program has been established at the Institute for Molecular Medicine Finland (FIMM), University of Helsinki, and the Helsinki University Hospital. Central to this program is drug sensitivity and resistance testing (DSRT), in which responses of primary cancer cells to a comprehensive clinical oncology and signal transduction drug collection are monitored. The drug sensitivity information is used with molecular profiling to establish hypotheses on individual cancer-selective targeting drug combinations and their predictive biomarkers, which can be explored in the clinic. Here, we describe how acoustic droplet ejection is enabling DSRT in our cancer individualized systems medicine program to (1) generate consistent but configurable assay-ready plates and determine how this affects data quality, (2) flexibly prepare drug combination testing plates, (3) dispense reagents and cells to the assay plates, and (4) perform ultra-miniaturized follow up assays on the cells from DSRT plates.
A handful of academic groups in Europe and the US are using high-throughput (HT) ex vivo screening to test the sensitivity of individual patient tumors to hundreds of combinations of cancer drugs - a strategy that in earlier iterations failed to predict response to therapy. HT screening is used routinely in drug discovery. Thanks to advances in nanoliter-scale sample handling and computational biology, evidence is slowly building the potential of HT approaches to identify novel combination therapies, first in leukemia and hopefully for treating other cancers.
The Malaria Box, assembled by the Medicines for Malaria Venture, is a set of 400 structurally diverse, commercially available compounds with demonstrated activity against blood-stage Plasmodium falciparum. The compounds are a representative subset of the 20,000 in vitro antimalarials identified from the high-throughput screening efforts of St. Jude Children's Research Hospital (TN, USA), Novartis and GlaxoSmithKline. In addition, a small set of active compounds from commercially available libraries was added to this group, but it has not previously been published. Elucidation of the biochemical pathways on which these compounds act is a major challenge; therefore, access to these compounds has been made available free of charge to the investigator community. Here, the Malaria Box compounds were tested for activity against the formation of b-hematin, a synthetic form of the heme detoxification biomineral, hemozoin. Further, the mechanism of action of these compounds within the malaria parasite was explored. Ten of the Malaria Box compounds demonstrated significant inhibition of b-hematin formation. In this assay, doseeresponse data revealed IC50 values ranging from 8.7 to 22.7 mM for these hits, each of which is more potent than chloroquine (a known inhibitor of hemozoin formation). The in vitro antimalarial activity of these ten hits was confirmed in cultures of the chloroquine sensitive D6 strain of the parasite resulting in IC50 values of 135e2165 nM, followed by testing in the multidrug resistant strain, C235. Cultures of P. falciparum (D6) were then examined for their heme distribution following treatment with nine of the commercially available confirmed compounds, seven of which disrupted the hemozoin pathway.
Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed.
Routine peptide structure-activity relationship screening requires the serial dilution of peptides to produce full concentration response curves. Established tip-based protocols involve multiple tip changes and high exposure to plasticware. In the case of peptides, this becomes a challenge, since peptides can adsorb to plastic, resulting in an observed loss of potency. Various methods can be employed to prevent peptide loss during compound handling, such as the inclusion of bovine serum albumin or solvents in assay buffer and the siliconization of plasticware, yet protein binding remains unpredictable. The degree of variation by which peptides will adhere to plasticware can confuse results and cause inaccuracies in potency predictions. We evaluated acoustic non-contact methods for peptide serial dilution and compared it with traditional tip-based methods, on the effect on potency curves for glucagon-like peptide-1 and glucagon peptide analogues. The current study demonstrates the benefits of non-contact dispensing for high-density microplate assay preparation of peptides using nanoliter droplets across our entire drug discovery workflow, from in vitro high-throughput screening to drug exposure determinations from in vivo samples.
Drug combination testing in the pharmaceutical industry has typically been driven by late-stage opportunistic strategies rather than by early testing to identify drug combinations for clinical investigation that may deliver improved efficacy. A rationale for combinations exists across a number of diseases in which pathway redundancy or resistance to therapeutics are evident. However, early assays are complicated by the absence of both assay formats representative of disease biology and robust infrastructure to screen drug combinations in a medium-throughput capacity. When applying drug combination testing studies, it may be difficult to translate a study design into the required well contents for assay plates because of the number of compounds and concentrations involved. Dispensing these plates increases in difficulty as the number of compounds and concentration points increase and compounds are subsequently rolled onto additional labware. We describe the development of a software tool, in conjunction with the use of acoustic droplet technology, as part of a compound management platform, which allows the design of an assay incorporating combinations of compounds. These enhancements to infrastructure facilitate the design and ordering of assay-ready compound combination plates and the processing of combinations data from high-content organotypic assays.
Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible highthroughput epigenetic screening. Echo liquid handlers can transfer compounds, samples, and reagents in sub-microliter volumes to high density assay formats using only acoustic energy - no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. The PHERAstar FS multi-mode plate reader, with the highest sensitivity and lowest read time of assays in high density plate formats, is a perfect complement to enable an unparalleled solution for cost-effective, high-throughput epigenetic screening. Using the HTRF EPIgeneous™ Methyltransferase kit from Cisbio, we developed a miniaturized methyltransferase assay that can be easily adapted to automation and increased throughput, while maintaining high data quality. HTRF assays are typically performed at volumes of about 20 µL in a 384-well low volume plate. However, with the nanoliter dispense increments of the Echo liquid handler, assay volumes can be reduced significantly while maintaining data quality. In this study we were able to reduce a methyltransferase assay to a 2 µL final volume with excellent results.
The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.
In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.
The high throughput direct measurement of substrate-to-product conversion by label-free detection has been considered the “Holy Grail” of drug discovery screening. Mass spectrometry as a detection system has the potential to be part of the ultimate screening solution. However, MS with existing sample introduction modes, despite being widely used in drug discovery typically cannot meet the throughput requirements of HTS. We propose a unique, innovative solution to the problem of throughput by using acoustic droplet ejection (ADE) to transfer femtoliter samples from microliter assays rapidly, precisely and accurately directly into a mass spectrometer. Acoustic technology has been widely used to support compound management activities within the pharmaceutical industry. The speed, accuracy, precision and robustness of acoustic dispensers have been proven. In principle, the integration of an acoustic source with a MS detector would result in a system capable of delivering ~4000 data points per hour into a high sensitivity label-free detector. It would enable sampling from 1536-well plates and reduce the total required assay volume to <5µL. The rapidity of sampling would enable real-time kinetic studies to capture multiple data points within the first minute of initiating a reaction. Together Labcyte Inc, Waters Corp and AstraZeneca have built a prototype acoustic source linked to a mass spectrometer. Droplets in the range of 50-200fL are acoustically ejected directly into the MS through a charge field. The ion beam is detected in the single quad MS where the typical signal has a very sharp attack profile and instant stop when the acoustic spray is turned on and off. This process produces a square wave signal which is simple to integrate for quantitative assays, and generates reliable and reproducible spectra. The system works in both positive and negative ion modes. The process is capable of producing singly or multiply charged species. The ability to load samples into a MS detector at such a high rate from much reduced assay volumes has significant potential not only within drug discovery but other areas of industry. Dynamic fluid analysis, the ability of the acoustic injector to adjust automatically to varying viscosities and surface tensions of the sample, allows the generation of droplets from a wide range of fluids including blood, plasma, cell culture medium, acid digests, and chemical syntheses. In principle, the simplicity of the acoustic source enables it to be fitted to any type of MS detector with an atmospheric pressure interface (single quad, triple quad, ToF), extending the range of applications into the “omics” field.