Phenotypic changes in biological function triggered by small molecules are at the core of drug discovery efforts internationally. Such alterations at the cellular level determine the direct effect of a bioactive compound on an advanced disease-relevant parameter. As a result, researchers are investigating compounds that affect pathophysiology in a target-agnostic manner.
Access to translationally relevant biological assays for large-scale screening and molecular target identification (or target deconvolution) based on the development of hits and leads represent two unique requirements of modern phenotypic-based drug discovery, according to Lorenz M. Mayr, vice president and global head, biological reagents and assay development at AstraZeneca.
More than 40 phenotypic assays based on high-content, flow cytometric, transcriptomic, reporter gene, and homogeneous time-resolved fluorescence (HTRF) approaches are available at AstraZeneca. A majority of them are amenable to high-throughput screening (i.e. 384-well format). Phenotypic biology as part of the early discovery approach is supported by fluorescent and brightfield microscopy, confocal and tissue imaging, live cell and kinetic platforms, image analytics, and data processing and management. High-content biology combines cellular imaging of complex biological systems with high-throughput techniques to allow rapid and disease-relevant cell screening.
Mayr explains that nearly 10% of all assays in the phenotypic assay library at AstraZeneca currently exist in a microphysiological system (MPS) format. Automated robust screening and analysis, and high-definition multiparametric assays are defining disease-relevant biological screening efforts previously restricted to mechanistic studies in target discovery.
”Gaining an understanding of the secretome function will lead to new opportunities for therapy.”
“The extracellular target space is a rich source of novel targets (with 70% of all approved drugs), but it remains largely unexplored, and holds significant value as part of phenotypic drug discovery,” says Mayr. ”Gaining an understanding of the secretome function will lead to new opportunities for therapy.” Researchers at AstraZeneca have used the rich secretome library of 6,400 proteins to identify relevant protein signals in disease. Human secretome-based small molecule drug screening in collaboration with KTH Stockholm has led to new treatments for post-myocardial infarction and heart failure.
Zebrafish chemical screens
Several therapeutically promising compounds originally identified in zebrafish chemical screens are at various stages of clinical development. Oricula Therapeutics is developing Proto-1 for prevention of hearing loss, Novo Biosciences is investigating a metalloproteinase-13 (MMP13) inhibitor for peripheral neuropathy, and La Jolla Pharmaceuticals is screening BMP receptor inhibitors for fibrodysplasia ossificans progressiva and other rare diseases.
A lack of understanding into the mechanism associated with inner ear hair cell death following aminoglycoside treatment had previously prevented therapeutic drug discovery, says Malcolm A. Gleser, CEO of Oricula Therapeutics. As Gleser explains, “the lateral line hair cells on the exterior of zebrafish offer an easily manipulable model for the phenotypic screening of hair cell toxins and protectants.” “The zebrafish screen not only has high sensitivity in detecting known ear toxins, but it identifies protectants that translate with high fidelity to mammalian models,” he adds.
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Leonard I. Zon, Grousbeck Professor of Pediatric Medicine at Harvard Medical School and director of the stem cell program at Boston Children's Hospital, says that “the zebrafish is a fantastic system for chemical genetics—you can simply add chemicals to water and screen for interesting phenotypes.” Researchers led by Zon found that prostaglandin E2 (PGE2) stimulated blood stem cells in the zebrafish embryo. “We then showed that the chemical works in a marrow transplant in mice, and proceeded to a Phase I trial involving 12 patients with leukemia.”
The investigators treated one of the cord blood samples with PGE2 for 2 hours and administered both cord blood units into the patient. “In 10 out of 12 patients, the treated cord blood unit predominated, and the neutrophils and platelets from that unit came back more quickly,” informs Zon. The Phase II results with 48 patients diagnosed with hematological malignancies and undergoing hematopoietic stem cell transplantation support the Phase I findings.
High-content screening of suspension cells
The phenotypic screening platform developed at Intellicyt enables high-end high-content screening (HCS) of suspension cells (such as immune cells), explains Angela K. Schultz, vice president, marketing. The platform offers multiplexing, miniaturization (using lower assay volumes ranging from 15 µL to 20 µL), and automation of arrays to screen large libraries formatted in 96-, 384- or 1536-well plates, explains Joseph M. Zock, senior director, product management. In fact, the analysis of a single plate in 384-well format is possible in 30 to 40 minutes, and the analysis of over a dozen plates can be accomplished in a single day. Multiplexing of cells and beads has been used to analyze checkpoint inhibitor antibodies for therapeutic drug discovery. The high-content assay enables rapid and simultaneous detection of multiple cytokines using only 30 µL of well volume.
Revvity uses confocal imagers for HCS “to generate tens to hundreds of independent measures at the single-cell level providing independent phenotypic information about the fate of the cell following drug or unknown chemical compound exposure,” according to O. Joseph Trask, senior application scientist, cellular imaging & analysis, discovery & analytical solutions. Proprietary HCS imaging platforms allow processing of up to 100,000 wells per day based on a single field per well with 4 independent fluorescent channels, equivalent to 400,000 images per field.
To facilitate phenotypic screening for small molecule drug discovery, Revvity has developed ultra-low attachment microplates for self-assembly of tumor cells into 3D cell models. Trask and colleagues screened a focused library of 3,000 small molecule compounds to identify and validate hits that promote the reversal of epithelial-mesenchymal transition in colorectal cancer. In collaboration with Daniel V. LaBarbera of the University of Colorado, Trask has screened a 3D organoid cancer cell model and validated 3D HCS algorithms using Revvity’s new Operetta CLS HCS imager to discover drug therapies effectively targeting the malignant phenotype.
High-throughput phenotypic screening for target deconvolution
Scientists at Charles River Laboratories have discovered 75 preclinical drug candidates over the past 17 years. The discovery is attributed to a streamlined process of integrated drug discovery of small molecules. According to David F. Fischer, executive director biology and DMPK Discovery, “Charles River has multiple examples where molecules identified during phenotypic screening went through successful target deconvolution at the hit-to-lead and lead-optimization stage.” The mechanism-of-action studies using adenoviral libraries and a strategic collaboration with Caprotec Bioanalytics contributed to successful target identification.
Using cell-based high-throughput screening of small molecules, the researchers tested different synthetic versions of the lead molecule O03 to identify a compound designated as BF844, with improved potency and pharmacokinetic features.1 BF844 stabilized the disease-causing mutation in a mouse model of Usher syndrome type III, which is associated with deafness, blindness, and balance disorders. “This molecule is obviously a first-in-class as this was the first mechanism ever explored for Usher type III with a small molecule,” Fischer says.
Charles River has “developed over 100 medium- and high-throughput customized cell-based assays for screening multiple disease indications in more than 25 human primary cell types, including those “derived from tissue, blood, and differentiated stem cells (iPSC and hESC).”
Indeed, phenotypic screening has the potential to discover novel therapeutic targets, which may have greater impact at the systems level than established targets. Target deconvolution and validation enhance the frequency of biologically active hits in phenotypic drug discovery.
References
1. Alagramam KN, Gopal SR, Geng R, et al. A small molecule mitigates hearing loss in a mouse model of Usher syndrome III. Nat Chem Biol. 2016;12(6):444-51. [PMID: 27110679]
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