November 24, 2021
To keep life sciences professionals abreast of the latest advances in assay development, the Assay Guidance Manual (AGM) offers an atlas to guide researchers through this crucial step of assay development for drug discovery. The December 2021 issues of the SLAS journals highlight ongoing AGM updates.
“The AGM captures best practices and technological advances to develop and implement robust assays in early translational research and to ensure that the right assays are developed for the right targets and biological processes,” says Sarine Markossian, Ph.D., editor-in-chief of the AGM and lead of the AGM translational science resources program at the National Center for Advancing Translational Sciences (NCATS, Rockville, MD, USA).
“Robust assays cultivate reproducibility – this is key to every successful and effective drug discovery campaign. Assays ride the pipeline of preclinical drug discovery from target development all the way to preclinical development,” Markossian continues. “We can’t stress more the importance of rigor in early drug discovery research. It helps accelerate the development of new therapies and reduces costs.”
Her AGM co-editor and senior advisor to the NCATS director, G. Sitta Sittampalam, Ph.D., agrees: “From the very beginning, you cannot move a molecule through a drug discovery program without robust assays,” he affirms. “The AGM focuses on the development of assays up front to identify pharmacologically and physiologically relevant molecules.”
Offering a wealth of techniques for optimizing assays with the overall goal of developing probes that modulate the activity of biological targets, pathways or cellular phenotypes, the AGM emerged from an internal training document at Eli Lilly and Company in the early 2000s.
“The company decided to release the document to the National Institutes of Health (NIH, Bethesda, MD, USA), with the advent of the NIH Molecular Libraries Program because they realized how important it is to drug discovery and development for published assays to be reproducible and replicable,” Sittampalam explains. “Articles from 10 years ago pointed out that much of the published research could not be reproduced – that is something the AGM continually tries to address with best practices assay development and data analysis.”
Now managed by NCATS, the AGM is a disease agnostic translational science program that benefits scientists in academic, non-profit, government and industrial research laboratories in the development of assay formats compatible with high-throughput screening (HTS) and Structure Activity Relationship (SAR) measurements of new and known molecular entities. The AGM is a free and publicly available eBook, and multiple workshops that are held virtually and across the country to help scientists design and implement robust assays in early translational research. The video recordings of those workshops are available online.
"The manual has more than 50 chapters and 13,000 printed pages – so you can see the depth and the breadth of its content,” Markossian says. She is excited to bring AGM topics into concurrent issues of SLAS Discovery and SLAS Technology for which she and Sittampalam serve as co-editors.
“We were asked to create these issues with topics that capture the assay guidance workshops that we present at the SLAS International Exhibition and Conference,” Markossian continues, adding that their goal is to expand on the lecture concepts by incorporating best practices in assay methodologies to enable reproducible results and illustrate how these principles are critical to the entire drug development process. The journal issues provide life sciences and technology investigators an introduction to the resources available in the AGM program and form a solid foundation for research.
“My hope is that these issues help scientists develop platforms for successful future drug discovery campaigns by following development and validation best practices that the AGM provides,” she says. “We need to continue an open dialog regarding best practices to avoid irreproducibility and increase efficiency in drug discovery. Life sciences needs to avoid spending billions of dollars on research that is not reproducible.”
Providing Best Practice Standards for Research
These SLAS journal issues mirror the AGM’s content, Markossian continues. “What takes center stage in this collection is best practices in assay development, analysis and implementation for HTS and lead discovery,” she explains, adding that six articles in the collection focus on specific approaches for success.
For example in the SLAS Discovery special issue, an article from Samarjit Patnaik, Ph.D., and colleagues from NCATS, “The Impact of Assay Design on Medicinal Chemistry: Case Studies,” shares studies from both NCATS Division of Preclinical Innovation (DPI) and the literature that examine how assay design and running multiple orthogonal assays improve medicinal chemistry efforts.
“The team reviews multiple case studies involving drug and chemical probe discovery in which the evaluation of compounds in secondary or orthogonal assays led to the discovery of unexpected activities. This forces a reevaluation of the original assay design that was used for the discovery of the compound’s biological activity,” Markossian says, adding that the authors emphasize early partnerships among assay biologists, informaticians and medicinal chemists.
“Patnaik’s article encourages chemists to cross boundaries toward an understanding of assay biology to better their medicinal chemistry efforts,” she says. This idea echoes an article published by colleagues at NCATS three years ago about how successful translational scientists are those who reach across boundaries early on in drug discovery.
The quest for best practices continues in an SLAS Discovery article from Terry Riss, Ph.D., and colleagues at Promega Corp. (Madison, WI, USA). “Treating Cells as Reagents to Design Reproducible Assays” describes how to establish proper standard procedures for cell culture preparation and handling.
“There are a lot of important details captured here,” Markossian comments. “The article includes resources for good cell culture practice and particularly emphasizes the importance of cell line authentication and determining cell number as an internal control.”
The importance of authentication is the focus of another SLAS Discovery article, “The Extensive and Expensive Impacts of HEp-2, Intestine 407, and Other False Cell Lines in Journal Publications” by author Christopher Korch, Ph.D., of the University of Colorado, Anschutz Divisions of Medical Oncology and Endocrinology (Aurora, CO, USA), and Amanda Capes-Davis, Ph.D., of the Children's Medical Research Institute, The University of Sydney (Westmead, NSW, Australia).
“This article highlights the problem of cell line misidentification and what the impact would be if left undetected,” Markossian says. “I think this is an important topic to discuss, and this article helps continue the dialog among scientists about this major problem. The article also provides tools, approaches and resources on how to address this issue.”
Other original research articles in SLAS Discovery include one from Jeffrey Weidner, Ph.D., QualSci Consulting, LLC, (Carmel, IN, USA) and colleagues, “Addressing Unusual Assay Variability with Robust Statistics,” which provides guidance on the use of varying statistical methods for the analysis of bioassay data as an alternative to standard approaches.
“In cases where assays do not conform to a normal distribution of errors, researchers must employ different statistics – using medians instead of mean, for example – so that outliers aren’t excluded, and the entire data set can be used,” says Sittampalam. “That’s one particular chapter that would be extremely useful to many scientists who are developing new assays that may exhibit non-gaussian data profiles.”
Another SLAS Discovery article examines nuisance compounds in “Addressing Compound Reactivity and Aggregation Assay Interferences: Case Studies of Biochemical High-Throughput Screening Campaigns Benefiting from the National Institutes of Health Assay Guidance Manual Guidelines.” Author Jayme Dahlin, M.D., Ph.D., from NCATS and colleagues, review two previously reported biochemical HTS campaigns for small-molecule inhibitors of the epigenetic targets Rtt109 and NSD2. The team assesses best practices and directs readers to high-yield resources in the AGM and elsewhere for the mitigation and identification of compound-dependent reactivity and aggregation assay interferences.
“There are cases in which you have to follow up on some of those nuisance compounds,” says Sittampalam. “You begin to wonder if the original assay was correct, or the compounds were bad in these circumstances. Researchers typically see unusual activity that doesn’t turn out to be right in subsequent assays.”
In “Busting Myths in Compound Handling Practices for Assay Developers,” which appears in the SLAS Technology special collection, Rosalia Gonzales, Ph.D., and fellow authors from Pfizer, Inc. (Groton, CT, USA), highlight recommended best practices that ensure sample integrity and present data that can help determine the root cause of an assay gone bad. The team shares how a strong, collaborative relationship between screening and sample handling groups leads to the discovery of breakthrough medicines.
Development and Validation
Another topic group addressed by the issues is assay development and validation, launched by an article in SLAS Discovery by Nathan Coussens, Ph.D., Frederick National Laboratory for Cancer Research, (Frederick, MD, USA) and colleagues. The article, “Complex Tumor Spheroids, a Tissue Mimicking Tumor Model, for Drug Discovery and Precision Medicine,” holds much potential, according to Markossian. The paper illustrates the importance of moving toward 3D tissue and other complex models in assay development for drug discovery for studying tumor response and mimicking the in vivo properties of drugs.
“Complex tumor spheroids incorporate human malignant and stromal cell components,” she explains. “The researchers behind this article developed and validated a robust complex tumor spheroid assay for HTS, and they implemented this assay with a variety of malignant pancreatic cell lines.”
“I’m excited about this article,” Markossian continues. “Physiologically relevant assays have great potential to better mimic the situation in vivo and capture proper biological activity of compounds.”
In the SLAS Discovery article “Validating ADME QSAR Models Using Marketed Drugs,” Pranav Shah, Ph.D., NIH, NCATS, and fellow authors explore NCATS’ Tier I ADME assays – kinetic aqueous solubility, the parallel artificial membrane permeability assay (PAMPA) and rat liver microsomal stability. Using recent data generated from in-house lead optimization from Tier I ADME studies to update QSAR models for these three endpoints, the team validates in silico performance against a set of marketed drugs.
“It is important to note that the authors make these models and data sets available online to the drug discovery community through the ADME@NCATS web portal,” Markossian comments.
From Meng Wu, Ph.D., of the University of Iowa High Throughput Screening Core, University of Iowa, (Iowa City, IA, USA) and colleagues, come lessons learned in the SLAS Discovery article, “A ‘Failed’ Assay Development for the Discovery of Rescuing Small Molecules from the Radiation Damage.” The team report that their discovery falls into “the proverbial valley of death” encountered in assay development, which includes phenotypic assays that aren’t exactly physiologically relevant and target-based assays that don’t necessarily correlate to phenotypic observations.
Rounding out the issues are articles that focus on the utility of specialized assays in lead discovery and target validation. Author Vance Lemmon, Ph.D., of the Miami Project to Cure Paralysis and University of Miami Miller School of Medicine, (Miami, FL, USA), and colleagues present “Phenotypic Screening Following Transcriptomic Deconvolution to Identify Transcription Factors Mediating Enhanced Axon Growth Induced by a Kinase Inhibitor.” This SLAS Discovery article explores a transcriptomic deconvolution of RO48-associated axon growth with the goal of identifying transcriptional regulators associated with axon growth in the central nervous system.
“This manuscript in a nutshell uses a powerful screening strategy to identify novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor,” says Markossian. “It’s a unique approach that identifies several potential outgrowth regulators.”
Another SLAS Discovery article, “Identification of Compounds for Butyrylcholinesterase (BChE) Inhibition,” from NCATS author Menghang Xia, Ph.D., and colleagues, examines a quantitative HTS approach to establish a robust and reliable process to screen large compound collections for potential BChE inhibitors – often regarded as a viable therapeutic approach in Alzheimer’s disease.
Two other articles that appear in SLAS Technology special collection describe emerging technologies related to HTS and lead optimization. In “Cross-Platform Bayesian Optimization System for Autonomous Biological Assay Development,” Dahlin and colleagues collaborate with an industrial partner, Kebotix (Cambridge, MA, USA), on remote-controlled autonomous bioassay optimization using Bayesian statistical approaches.
“An NCATS team demonstrates cloud-based assay development remotely, running assays in a Boston facility so that they can optimize the assays much more quickly using robots. This is a very new area of study coming up quickly,” says Sittampalam.
In “Automated High-Throughput System Combining Small Scale Synthesis with Bioassays and Reaction Screening,” a perspective article in SLAS Technology, author R. Graham Cooks, Ph.D., Purdue University (West Lafayette, IN, USA) and colleagues describe exciting new applications of desorption electrospray ionization (DESI) mass spectrometry (MS) for high-throughput synthetic chemistry, chemical reaction screening and label-free biological assays that are amenable to HTS.
“This research uses an unconventional approach to not only screen compounds being made but also to test them in a mass spec assay to see whether these compounds are active,” describes Sittampalam, who hopes that more AGM-relevant articles featuring breakthroughs in instrumentation and automation can be featured in upcoming issues of SLAS Technology.
“I am quite excited about some of these topic areas and what these new chapters hold,” Sittampalam concludes. “The AGM covers not only best practices, but also new technologies and how to use existing technologies to accelerate drug discovery.”