Perspectives on phenotypic screening-Screen Design and Assay Technology Special Interest Group.

Here we offer perspectives on phenotypic screening based on a wide-ranging discussion entitled "Phenotypic screening, target ID, and multi-omics: enabling more disease relevance in early discovery?" at the Screen Design and Assay Technology Special Interest Group Meeting at the 2023 SLAS Conference. During the session, the authors shared their own experience from within their respective organizations, followed by an open discussion with the audience. It was recognized that while substantial progress has been made towards translating disease-relevant phenotypic early discovery into clinical success, there remain significant operational and scientific challenges to implementing phenotypic screening efforts, and improving translation of screening hits comes with substantial resource demands and organizational commitment.

Roadmap for the use of base editors to decipher drug mechanism of action.

CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors.

Hit Triage and Validation in Phenotypic Screening: Considerations and Strategies.

The promise of phenotypic screening resides in its track record of novel biology and first-in-class therapies. However, challenges stemming from major differences between target-based and phenotypic screening do exist. These challenges prompted us to rethink the critical stage of hit triage and validation on the road to clinical candidates and novel drug targets.

Challenges and Opportunities in Enabling High-Throughput, Miniaturized High Content Screening.

Within the Drug Discovery industry, there is a growing recognition of the value of high content screening (HCS), particularly as researchers aim to screen compounds and identify hits using more physiologically relevant in vitro cell-based assays. Image-based high content screening, with its combined ability to yield multiparametric data, provide subcellular resolution, and enable cell population analysis, is well suited to this challenge. While HCS has been in routine use for over a decade, a number of hurdles have historically prohibited very large, miniaturized high-throughput screening efforts with this platform.

Generation of a clonal induced pluripotent stem cell (iPSC) line expressing the mutant MECP2 allele from a Rett Syndrome patient fibroblast line.

Human fibroblast cells collected from a 3-year old, female Rett Syndrome patient with a 32bp deletion in the X-linked MECP2 gene were obtained from the Coriell Institute. Fibroblasts were reprogrammed to iPSC cells using a Sendai-virus delivery system expressing human KOSM transcription factors. Cell-line pluripotency was demonstrated by gene expression, immunocytochemistry, in-vitro differentiation trilineage capacity and was of normal karyotype.

Dimethadione embryotoxicity in the rat is neither correlated with maternal systemic drug concentrations nor embryonic tissue levels.

Pregnant rats treated with dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, produce offspring having a 74% incidence of congenital heart defects (CHD); however, the incidence of CHD has high inter-litter variability (40-100%) that presents a challenge when studying the initiating events prior to the presentation of an abnormal phenotype. We hypothesized that the variability in CHD incidence was the result of differences in maternal systemic concentrations or embryonic tissue concentrations of DMO. To test this hypothesis, dams were administered 300 mg/kg DMO every 12h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses).

Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists.

G protein-coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment.

Establishing a High-content Analysis Method for Tubulin Polymerization to Evaluate Both the Stabilizing and Destabilizing Activities of Compounds.

Microtubules are important components of the cellular cytoskeleton that play roles in various cellular processes such as vesicular transport and spindle formation during mitosis. They are formed by an ordered organization of α-tubulin and β-tubulin hetero-polymers. Altering microtubule polymerization has been known to be the mechanism of action for a number of therapeutically important drugs including taxanes and epothilones.

Noninvasive high-resolution ultrasound reveals structural and functional deficits in dimethadione-exposed fetal rat hearts in utero.

Background: We previously showed dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, induces ventricular septation defects (VSD) and other heart anomalies in rat (Weston et al., 2011). Because of the relationship between cardiac structure and function, we hypothesized that DMO-induced structural defects of the heart are associated with in utero functional deficits.

Functional deficits among patients with mild cognitive impairment.

Background: Diagnostic criteria for mild cognitive impairment (MCI) include no significant functional decline, but recent studies have suggested that subtle deficits often exist. It is not known whether these differ by MCI type. We investigated the level and type of functional impairment among patients with MCI.

Co-variation in frequency and severity of cardiovascular and skeletal defects in Sprague-Dawley rats after maternal administration of dimethadione, the N-demethylated metabolite of trimethadione.

Background: The anticonvulsant trimethadione is a potent inducer of ventricular septation defects, both clinically and in rodents. Teratogenicity requires its N-demethylation to dimethadione, the proximate teratogen. It was previously demonstrated trimethadione only induced membranous ventricular septation defects in rat (Fleeman et al.

Association of plasma beta-amyloid level and cognitive reserve with subsequent cognitive decline.

Context: Lower plasma β-amyloid 42 and 42/40 levels have been associated with incident dementia, but results are conflicting and few have investigated cognitive decline among elders without dementia.

Objective: To determine if plasma β-amyloid is associated with cognitive decline and if this association is modified by measures of cognitive reserve.

Design, Setting, And Participants: We studied 997 black and white community-dwelling older adults from Memphis, Tennessee, and Pittsburgh, Pennsylvania, who were enrolled in the Health ABC Study, a prospective observational study begun in 1997-1998 with 10-year follow-up in 2006-2007.

Factors associated with hepatitis A vaccination receipt in one-year-olds in the state of Michigan.

The objectives of this study were to determine factors associated with hepatitis A vaccination and to assess overall hepatitis A vaccination coverage levels among one-year-olds in Michigan. The study population was the first hepatitis A vaccination-eligible birth cohort (n = 134 226) enrolled in the Michigan Care Improvement Registry (MCIR) after 2006 recommendations were made to routinely vaccinate all one-year-olds. All children whose first birthday occurred on or between May 1, 2006 and April 31, 2007 were included in the study population.

Potentially inappropriate medication use in older adults with mild cognitive impairment.

Background: Patients with mild cognitive impairment (MCI) may be especially vulnerable to the side effects of potentially inappropriate medications (PIMs), especially those that impair cognition.

Methods: We conducted a cross-sectional study to determine the prevalence of PIM use among 689 patients with MCI. We used the 2003 Beers Criteria for cognitive impairment to identify PIMs.

The metabolic syndrome and development of cognitive impairment among older women.

Background: Several studies support a role for cardiovascular risk factors in cognitive aging. The metabolic syndrome, a constellation of cardiovascular risk factors, is common in elderly people. A growing but conflicting body of literature suggests that the metabolic syndrome may be associated with cognitive impairment.

Thoracic skeletal defects and cardiac malformations: a common epigenetic link?

Congenital heart defects (CHDs) are the most common birth defects in humans. In addition, cardiac malformations represent the most frequently identified anomaly in teratogenicity experiments with laboratory animals. To explore the mechanisms of these drug-induced defects, we developed a model in which pregnant rats are treated with dimethadione, resulting in a high incidence of heart malformations.

A role for cyclic nucleotide monophosphates in synaptic modulation by a crayfish neuropeptide.

DF2 (DRNFLRFamide), a FMRFamide-like peptide, has been shown to increase the amount of transmitter released at crayfish neuromuscular junctions. Here, we examined a possible role for the cyclic nucleotide monophosphates, cAMP and cGMP, in DF2's effects on synaptic transmission. The effects of DF2 on synaptic transmission were monitored by recording excitatory postsynaptic potentials (EPSPs) in the deep abdominal extensor muscles of the crayfish, Procambarus clarkii.

A data integration methodology for systems biology.

Different experimental technologies measure different aspects of a system and to differing depth and breadth. High-throughput assays have inherently high false-positive and false-negative rates. Moreover, each technology includes systematic biases of a different nature.

A data integration methodology for systems biology: experimental verification.

The integration of data from multiple global assays is essential to understanding dynamic spatiotemporal interactions within cells. In a companion paper, we reported a data integration methodology, designated Pointillist, that can handle multiple data types from technologies with different noise characteristics. Here we demonstrate its application to the integration of 18 data sets relating to galactose utilization in yeast.

Systems biology, proteomics, and the future of health care: toward predictive, preventative, and personalized medicine.

The emergence of systems biology is bringing forth a new set of challenges for advancing science and technology. Defining ways of studying biological systems on a global level, integrating large and disparate data types, and dealing with the infrastructural changes necessary to carry out systems biology, are just a few of the extraordinary tasks of this growing discipline. Despite these challenges, the impact of systems biology will be far-reaching, and significant progress has already been made.

Revisiting the role of retinoid signaling in skeletal development.

Several years ago, it was discovered that an imbalance of vitamin A during embryonic development has dramatic teratogenic effects. These effects have since been attributed to vitamin A's most active metabolite, retinoic acid (RA), which itself profoundly influences the development of multiple organs including the skeleton. After decades of study, researchers are still uncovering the molecular basis whereby retinoids regulate skeletal development.

Inhibition of p38 MAPK signaling promotes late stages of myogenesis.

Signaling through the p38 mitogen-activated protein kinases (MAPKs) is essential for cartilage formation in primary cultures of limb mesenchyme. Here we show that, concurrent with a decrease in chondrogenesis, inhibition of p38 in limb bud cultures dramatically promotes muscle development. Specifically, treatment of primary limb bud cultures with p38 inhibitors increases the expression of myogenic markers and causes a striking increase in formation of myotubes, which were detected using antibodies specific for myosin heavy chain.

Molecular mechanisms regulating chondroblast differentiation.

Background: Formation of the cartilage template involves a multi-step process in which prechondrogenic mesenchymal cells form condensations prior to differentiating into matrix-producing chondroblasts. Retinoids, particularly retinoic acid, are among the numerous signaling molecules that have been implicated in this process. A proper balance of retinoids is essential for normal skeletal development in that too much or too little negatively impacts skeletogenesis.

Active repression by unliganded retinoid receptors in development: less is sometimes more.

The retinoid receptors have major roles throughout development, even in the absence of ligand. Here, we summarize an emerging theme whereby gene repression, mediated by unliganded retinoid receptors, can dictate cell fate. In addition to activating transcription, retinoid receptors actively repress gene transcription by recruiting cofactors that promote chromatin compaction.

Requirement for RAR-mediated gene repression in skeletal progenitor differentiation.

Chondrogenesis is a multistep process culminating in the establishment of a precisely patterned template for bone formation. Previously, we identified a loss in retinoid receptor-mediated signaling as being necessary and sufficient for expression of the chondroblast phenotype (Weston et al., 2000.