ABSTRACT
First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Patricia Dranchak is first author on ‘ In vivo quantitative high-throughput screening for drug discovery and comparative toxicology’, published in DMM. She is a staff scientist in the lab of James Inglese at the National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA. Patricia is interested in developing and integrating novel strategies and model systems to identify chemical modulators targeting the molecular basis of rare and neglected disease pathophysiologies.
Patricia Dranchak
How would you explain the main findings of your paper to non-scientific family and friends?
C. elegans is a soil-dwelling round worm that has been used by biologists in labs for decades, even contributing to a few Nobel Prizes! Advances in molecular biology can now endow organisms with the same gene mutations responsible for human diseases. When the organism is expressing fluorescently labelled proteins they can be detected and quantified by detectors used in laboratories carrying out high-throughput screening (HTS). Here, we have created a means to use such genetically modified nematodes to identify drugs that have the potential to help the study and treatment of rare, and not-well-understood human disorders.
What are the potential implications of these results for your field of research?
Pre-clinical drug discovery translational research can focus heavily on target-based and cellular model systems, partly, due to assay compatibility with high-throughput compound screening. Although important and effective, these assay designs can overlook unknown biological mechanisms of action and limit the information acquired from the primary HTS, including compound bioavailability and systemic toxicity. These assays might require several iterations by using orthogonal assays to eliminate false positives and prioritize active putative lead molecules. The application of whole-organism quantitative HTS (qHTS) using C. elegans can enable phenotypic assays based on complex genetic settings, and include pharmacological and absorption, distribution, metabolism and elimination (ADME) toxicological assessments of compounds from the initial screen. To date, most HTS efforts using this nematode have involved microscopy-based high-content imaging (HCI). However, although sensitive and phenotypically informative, HCI can be time consuming and computationally intensive; thus, primary screenings of large libraries of compounds are often assayed at a single concentration. The use of laser scanning cytometry (LSC) with C. elegans grown on the novel non-replicating food source of empty cell envelopes derived from E. coli, i.e. E. coli ghosts, offer an opportunity to study the phenology of various human diseases in a highly efficient and quantifiable manner over several generations. Our approach will enable the use of qHTS, during which compounds are tested over a wide concentration range to provide pharmacological data, including the EC50 of a dose response that may be suited to drug repurposing studies.
What are the main advantages and drawbacks of the experimental system you have used as it relates to the disease you are investigating?
C. elegans are an easy laboratory specimen to grow and maintain. To generate thermally induced E. coli ghosts as a novel non-replicating food source is an easy and cost-effective method to generate a nutrient medium for large-scale screening using worms. The rapid plate reading time and on-the-fly data analysis of several phenotypic parameters – of up to three different fluorescent channels across an assay plate by using LSC – also adds a new level of efficiency regarding data acquisition obtained with qHTS. However, based on their size and life span, HTS efforts with this organism are limited to a 384-well microtiter plate format and, depending on the phenotype of interest, time courses can persist for more than a week. Appropriate strains of worms, either with a phenotypic disease homolog of interest or humanized pathophysiological strains, require some expertise to generate and optimize them for phenotypic screening.
“In less than 30 min I had developed a protocol […] to identify not only the number of worms but also the body area, fluorescent intensity and general life stage of each object in the well,…”
The C. elegans in vivo quantitative high-throughput screening workflow (top left to bottom right). Fluorescently labelled worms modelling a biology of interest are dispensed in 384-well microtiter plate format amenable to compound treatment across a pharmacologically relevant concentration range. Laser-scanning cytometry is then used to quantify phenotypes of interest allowing dose-response curves to be plotted from this primary screening output. Correlation of biological response across chemotypes facilitates structure activity relationships, and candidate lead molecules can be further investigated with phenotypic and omics-based follow-up studies. Illustration by J. Inglese using BioRender.
The C. elegans in vivo quantitative high-throughput screening workflow (top left to bottom right). Fluorescently labelled worms modelling a biology of interest are dispensed in 384-well microtiter plate format amenable to compound treatment across a pharmacologically relevant concentration range. Laser-scanning cytometry is then used to quantify phenotypes of interest allowing dose-response curves to be plotted from this primary screening output. Correlation of biological response across chemotypes facilitates structure activity relationships, and candidate lead molecules can be further investigated with phenotypic and omics-based follow-up studies. Illustration by J. Inglese using BioRender.
What has surprised you the most while conducting your research?
Our model organism continues to amaze me with its flexibility and use for disease modeling. When I loaded the first 384-well plate of GFP-expressing C. elegans into the laser scanning cytometer, I was unsure as to what to expect. Being able to visualize individual worms, GFP distribution throughout the body during various life-stages, and shape and morphology of individual C. elegans within an individual well was exciting. In less than 30 min I had developed a protocol on the cytometer to identify not only the number of worms but also the body area, fluorescent intensity and general life stage of each object in the well, while eliminating background fluorescence. This technology has great potential for analyzing and quantifying various phenotypic homologs for human disease in the worm in ∼20% of the time it takes to read an equivalent plate on the high-content imager.
What do you think is the most significant challenge impacting your research at this time and how will this be addressed over the next 10 years?
Currently, I run our C. elegans screening platform with assistance from a research technician as part of a larger laboratory effort in pre-clinical translation. This limits the number of projects we can incorporate with this paradigm. I hope to expand our project portfolio for this model system, and would welcome collaborations and trainees at all levels (e.g. post-baccalaureate intramural research training awards or postdocs) interested in developing a translational research project by using this model organism. Also, the challenge I see is to establish compelling studies with C. elegans, demonstrating its power when it comes to advancing preclinical drug discovery. Ideally, we would be able to integrate this platform with the existing technology of the ADST lab (https://www.youtube.com/watch?v=VxtpySnbX-c) and the National Center for Advancing Translational Sciences (NCATS).
What changes do you think could improve the professional lives of scientists?
Science evolved to be more interdisciplinary, making broad collaborations both crucial and necessary to the progress of today's research efforts. Encouraging open access, having greater opportunities for data and, importantly, protocol sharing across labs and institutions is a worthwhile initiative. Building on this free exchange of information, access to varying scientific expertise and technology through collaborative endeavors will facilitate more-efficient program advancements. Identifying and accessing institutions with the ability to develop interdisciplinary partnerships across various research areas is imperative to enable academia to solve important biomedical problems. The C. elegans scientific community is highly organized, collaborative and open. The establishment and maintenance of the Caenorhabditis Genetics Center (CGC) with its repository of various strains of worms and the existence of WormBase are examples of how this research community is promoting open access and collaboration. The NIH and, specifically, the NCATS, offer unique opportunities to network additional expertise, such as assay development, medicinal chemistry, proteomics and large-scale data analysis – all of which can drive multidisciplinary collaborations.
What's next for you?
I am working to further develop this model organism screening platform here at the NCATS. We are looking to expand our operations by seeking new collaborations with other labs that use C. elegans models for various diseases, to carry out either focused or high-impact compound library screening. Towards this effort, we also look to expand our partnership with chemists who develop high-impact small-molecule, peptide or natural-product libraries, to enable screening across different assay systems. In addition to the potential breadth of human disease phenotypes that can be modeled with C. elegans, we are interested in using the qHTS platform to identify those that might be candidates for drug-repurposing studies.
Patricia Dranchak’s contact details: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Dr., Rockville, MD 20877, USA.
E-mail: [email protected]
