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. This Perspective assesses progress, highlights pitfalls, and offers possible solutions to help unlock the therapeutic potential of phenotypic drug discovery. Areas explored comprise screening and hit validation strategy, choice of cellular model, moving beyond 2D cell culture into three dimensions, and leveraging high-dimensional data sets downstream of phenotypic screens.

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. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The ß2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.

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. Whereas this process is usually straightforward for target screening hits, phenotypic screening hits act through a variety of mostly unknown mechanisms within a large and poorly understood biological space. Our analysis suggests successful hit triage and validation is enabled by three types of biological knowledge-known mechanisms, disease biology, and safety-while structure-based hit triage may be counterproductive.

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. Suitable hardware and consumables for conducting 1536-well HCS have only recently become available, and developing a reliable informatics framework to accommodate the scale of high-throughput HCS data remains a considerable challenge. Additionally, innovative approaches are needed to interpret the large volumes of content-rich information generated. Despite these hurdles, there has been a growing interest in screening large compound inventories using this platform. Here, we outline the infrastructure developed and applied at Bristol-Myers Squibb for 1536-well high content screening and discuss key lessons learned.

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. Interestingly, subsequent clones retained the epigenetic memory of the parent fibroblasts allowing for the segregation of wild-type and mutant expressing clones. This MECP2 mutant expressing clone may serve as a model for investigating MECP2 reactivation in Rett's Syndrome.

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). Maternal serum levels of DMO were assessed on GD 11, 12, 13, 14, 15, 18 and 21. Embryonic tissue concentrations of DMO were assessed on GD 11, 12, 13 and 14. In a separate cohort of GD 12 embryos, DMO concentrations and parameters of growth and development were assessed to determine if tissue levels of DMO were correlated with these endpoints. Embryos were exposed directly to different concentrations of DMO with whole embryo culture (WEC) and their growth and development assessed. Key findings were that neither maternal systemic concentrations nor tissue concentrations of DMO identified embryos that were sensitive or resistant to DMO in vivo. Direct exposure of embryos to DMO via WEC also failed to show correlations between embryonic concentrations of DMO with developmental outcomes in vitro. We conclude that neither maternal serum nor embryonic tissue concentrations of DMO predict embryonic outcome.

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. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor ß-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of ß-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.

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 ß-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. Traditional cell-based assays for tubulin-interacting compounds rely on their indirect effects on cell cycle and/or cell proliferation. Direct monitoring of compound effects on microtubules is required to dissect detailed mechanisms of action in a cellular setting. Here we report a high-content assay platform to monitor tubulin polymerization status by directly measuring the acute effects of drug candidates on the cellular tubulin network with the capability to dissect the mechanisms of action. This high-content analysis distinguishes in a quantitative manner between compounds that act as tubulin stabilizers versus those that are tubulin destabilizers. In addition, using a multiplex approach, we expanded this analysis to simultaneously monitor physiological cellular responses and associated cellular phenotypes.

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. To test the hypothesis, the goals were (1) define the parameters for ultrasound in the rat conceptus, and; (2) use ultrasound to identify structural and functional deficits following DMO treatment. METHODS: Different ultrasound modes (B-mode, M-mode, and Pulse-wave Doppler) using four high-resolution ultrasound transducer heads of varying frequency (25-40 MHz) were tested on gestational day (GD) 14, 15, 16, 17, and 21. Having identified the optimal conditions, pregnant Sprague-Dawley rats were administered six 300 mg/kg doses of DMO every 12 hr beginning at 19:00 hr on GD 8 to generate conceptuses with a high incidence of VSD. RESULTS: The three ultrasound modalities were used to identify VSD and several novel and rare structural heart anomalies (cardiac effusions and bifurcated septum) in live rat fetuses. DMO-treated hearts had an array of functional deficits including a decrease in mean heart rate, ejection fraction, and cardiac output and increased incidence of bradycardia and dysrhythmia. CONCLUSIONS: The ultrasound biomicroscope is an effective tool for the real-time characterization of the structure and function of embryo/fetal rat hearts. DMO causes significant deficits to in utero heart function for up to ten days (GD 21) following its final administration, suggesting long-term or possible permanent changes cardiac function.

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. METHODS: We studied 498 patients, evaluated at the Alzheimer's Disease Research Centers of California between 2006 and 2009, who had multidisciplinary evaluations by experts, including neurologic examination and neuropsychological testing. Patients were diagnosed with MCI and subtype was determined using cognitive domain scores. In a cross-sectional descriptive study, we examined whether functional impairment differed by MCI subtype, using the Blessed Roth Dementia Rating Scale (range: 0-17, higher scores indicating more impairment). RESULTS: Among the participants, the mean age was 75.4 years, 50.7% were women, and 81.7% were white. Patients with amnestic- (n = 392, 78.7%) and nonamnestic-type (n = 106, 21.3%) MCI had similar total Blessed Roth Dementia Rating Scale (1.6 and 1.5, respectively; P = .84) and Mini-Mental State Examination (26.5 and 26.7, respectively; P = .60) scores. Patients with amnestic MCI were more likely to have difficulty in remembering lists and recalling recent events (P < .05 for both) and less likely to have difficulty in eating and with continence (P = .01 for both), as compared with those with nonamnestic MCI. CONCLUSIONS: Despite the MCI diagnostic criteria suggesting no functional impairment, our results indicate that patients with MCI experience mild functional deficits that vary according to the type of 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., 2004), and our present goal is to determine whether direct administration of dimethadione increases the incidence and severity of septation defects. METHODS: Pregnant Sprague-Dawley rats were divided into five groups and administered either distilled water (control) or four different regimens of dimethadione. The core treatment was 300 mg/kg dimethadione b.i.d. on gestation day 9, 10 with additional groups given one additional dose of dimethadione 12 hr earlier, 12 hr later or two additional doses 12 hr earlier and later. Caesarian sections occurred on gestation day 21 and fetuses were examined for standard developmental toxicity endpoints. RESULTS: The broadest dosing regimen yielded the highest incidence and the most severe heart and axioskeletal findings with a decrease in mean fetal body weight. The overall incidence of ventricular septation defects was 74%, of which 68% were membranous and 9% muscular. Outflow tract anomalies (17%) were also observed, as were malformations of the axioskeleton (97%), but not of the long bones, and of particular interest was the high incidence of sternoschesis. CONCLUSIONS: Unlike trimethadione, dimethadione induces more serious muscular septation defects that are believed to be more clinically relevant. This, when taken together with the high incidence of total septation anomalies suggests dimethadione is useful for the study of chemically induced ventricular septation defects.

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

CONTEXT: Lower plasma ß-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 ß-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. Participant mean age was 74.0 (SD, 3.0) years; 55.2% (n = 550) were female; and 54.0% (n = 538) were black. MAIN OUTCOME MEASURES: Association of near-baseline plasma ß-amyloid levels (42 and 42/40 measured in 2010) and repeatedly measured Modified Mini-Mental State Examination (3MS) results. RESULTS: Low ß-amyloid 42/40 level was associated with greater 9-year 3MS cognitive decline (lowest ß-amyloid tertile: mean change in 3MS score, -6.59 [95% confidence interval [CI], -5.21 to -7.67] points; middle tertile: -6.16 [95% CI, -4.92 to -7.32] points; and highest tertile: -3.60 [95% CI, -2.27 to -4.73] points; P < .001). Results were similar after multivariate adjustment for age, race, education, diabetes, smoking, and apolipoprotein E [APOE ] e4 status and after excluding the 72 participants with incident dementia. Measures of cognitive reserve modified this association whereby among those with high reserve (at least a high school diploma, higher than sixth-grade literacy, or no APOE e4 allele), ß-amyloid 42/40 was less associated with multivariate adjusted 9-year decline. For example, among participants with less than a high school diploma, the 3MS score decline was -8.94 (95% CI, -6.94 to -10.94) for the lowest tertile compared with -4.45 (95% CI, -2.31 to -6.59) for the highest tertile, but for those with at least a high school diploma, 3MS score decline was -4.60 (95% CI,-3.07 to -6.13) for the lowest tertile and -2.88 (95% CI,-1.41 to -4.35) for the highest tertile (P = .004 for interaction). Interactions were also observed for literacy (P = .005) and for APOE e4 allele (P = .02). CONCLUSION: Lower plasma ß-amyloid 42/40 is associated with greater cognitive decline among elderly persons without dementia over 9 years, and this association is stronger among those with low measures of cognitive reserve.

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. Racial/ethnic minorities had increased odds of receiving the hepatitis A vaccination in Michigan, and Medicaid and WIC status modified this relationship. Fully understanding these relationships will be useful in targeting vaccination outreach and education programs.

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. We then determined if certain patients were more likely to use PIMs. RESULTS: There were 143 (20.8%) patients with MCI taking a PIM: 108 (15.7%) patients were taking one PIM and 35 (5.1%) patients were taking two or more PIMs. The most common PIMs were anticholinergics (35.7%) and benzodiazepines (31.5%). Patients were more likely to be taking PIMs if they were women and were taking a greater number of medications and less likely if they had a history of myocardial infarction. CONCLUSIONS: Patients with MCI are frequently taking PIMs that may negatively affect cognition. Future research is needed to assess whether cognitive impairment symptoms are improved if PIM use is reduced.

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. OBJECTIVE: To investigate the association between the metabolic syndrome and its components and incident cognitive impairment in older women. DESIGN: We prospectively determined if the metabolic syndrome and its components were associated with a 4-year risk of developing cognitive impairment (dementia, mild cognitive impairment, or low global cognitive test score). SETTING: The study was conducted at 180 clinical centers in 25 countries. PARTICIPANTS: A total of 4895 older women (mean age, 66.2 years) with osteoporosis who were part of an ancillary study to determine clinically relevant cognitive impairment were included in this study. These women were free of baseline cognitive impairment and had metabolic syndrome component measures. MAIN OUTCOME MEASURES: Clinically significant cognitive impairment was defined to include women with clinically adjudicated dementia or MCI and women who had a Short Blessed test score greater than 6 (consistent with impairment), but whose cases were not clinically adjudicated. Logistic regression analysis was used to examine the association between presence of the metabolic syndrome and development of clinically significant cognitive impairment. RESULTS: A total of 497 women (10.2%) had the metabolic syndrome and, of these, 36 (7.2%) developed cognitive impairment compared with 181 (of 4398 or 4.1%) without the syndrome (age-adjusted odds ratio, 1.66; 95% confidence interval, 1.14-2.41). The mean (SD) number of metabolic syndrome components for all women was 1.0 (1.1); 518 women (10.6%) were obese, 895 (18.3%) had hypertriglyceridemia, 1200 (24.5%) had low high-density lipoprotein cholesterol levels, 1944 (39.7%) had high blood pressure, and 381 (7.8%) had high fasting blood glucose levels. There was a 23.0% age-adjusted increase in the risk of developing cognitive impairment (odds ratio, 1.23; 95% confidence interval, 1.09-1.39) per unit increase in the number of components. Further multivariable adjustment somewhat reduced the effect. CONCLUSION: We found an association between the metabolic syndrome and the number of components and risk of developing cognitive impairment in older women. Additional studies are needed to determine if screening and close management of these at-risk elderly women would diminish the incidence of cognitive impairment.

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 number of activators and inhibitors were used to determine whether or not cAMP, cGMP, protein kinase A (PKA) and protein kinase G (PKG) mediate the effect of this neuropeptide. Phosphodiesterase inhibitors, known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of DF2 on synaptic transmission. Activators of PKA (Sp-cAMPS) and PKG (8-pCPT-cGMP) increase EPSP amplitude, mimicking the effects of DF2. Inhibitors of PKA (Rp-cAMPS) and PKG (Rp-8-pCPT-cGMPS) each block a portion of the response to the peptide, and when applied together these two inhibitors completely block the response. Taken together, these results indicate that cyclic nucleotides and cyclic nucleotide-dependent protein kinases are necessary components of the pathway underlying modulation by this neuropeptide.

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. Interestingly, these heart defects were accompanied by thoracic skeletal malformations (cleft sternum, fused ribs, extra or missing ribs, and/or wavy ribs), which are characteristic of anterior-posterior (A/P) homeotic transformations and/or disruptions at one or more stages in somite development. A review of other teratogenicity studies suggests that the co-occurrence of these two disparate malformations is not unique to dimethadione, rather it may be a more general phenomenon caused by various structurally unrelated agents. The coexistence of cardiac and thoracic skeletal malformations has also presented clinically, suggesting a mechanistic link between cardiogenesis and skeletal development. Evidence from genetically modified mice reveals that several genes are common to heart development and to formation of the axial skeleton. Some of these genes are important in regulating chromatin architecture, while others are tightly controlled by chromatin-modifying proteins. This review focuses on the role of these epigenetic factors in development of the heart and axial skeleton, and examines the hypothesis that posttranslational modifications of core histones may be altered by some developmental toxicants.

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. These data include global changes in mRNA and protein abundance, genome-wide protein-DNA interaction data, database information, and computational predictions of protein-DNA and protein-protein interactions. We divided the integration task to determine three network components: key system elements (genes and proteins), protein-protein interactions, and protein-DNA interactions. Results indicate that the reconstructed network efficiently focuses on and recapitulates the known biology of galactose utilization. It also provided new insights, some of which were verified experimentally. The methodology described here, addresses a critical need across all domains of molecular and cell biology, to effectively integrate large and disparate data sets.

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. These differences make network reconstruction from multiple data sets difficult and error-prone. Additionally, because of the rapid rate of progress in biotechnology, there is usually no curated exemplar data set from which one might estimate data integration parameters. To address these concerns, we have developed data integration methods that can handle multiple data sets differing in statistical power, type, size, and network coverage without requiring a curated training data set. Our methodology is general in purpose and may be applied to integrate data from any existing and future technologies. Here we outline our methods and then demonstrate their performance by applying them to simulated data sets. The results show that these methods select true-positive data elements much more accurately than classical approaches. In an accompanying companion paper, we demonstrate the applicability of our approach to biological data. We have integrated our methodology into a free open source software package named POINTILLIST.

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. Moving forward, the issue of how to use systems biology to improve the health of individuals must be a priority. It is becoming increasingly apparent that the field of systems biology and one of its important disciplines, proteomics, will have a major role in creating a predictive, preventative, and personalized approach to medicine. In this review, we define systems biology, discuss the current capabilities of proteomics and highlight some of the necessary milestones for moving systems biology and proteomics into mainstream health care.