Biochemical and Structural Characterization of a Peptidic Inhibitor of the YAP:TEAD Interaction That Binds to the α-Helix Pocket on TEAD.

The TEAD transcription factors are the most distal elements of the Hippo pathway, and their transcriptional activity is regulated by several proteins, including YAP. In some cancers, the Hippo pathway is deregulated and inhibitors of the YAP:TEAD interaction are foreseen as new anticancer drugs. The binding of YAP to TEAD is driven by the interaction of an α-helix and an Ω-loop present in its TEAD-binding domain with two distinct pockets at the TEAD surface. Using the mRNA-based display technique to screen a library of in vitro-translated cyclic peptides, we identified a peptide that binds with a nanomolar affinity to TEAD. The X-ray structure of this peptide in complex with TEAD reveals that it interacts with the α-helix pocket. Under our experimental conditions, this peptide can form a ternary complex with TEAD and YAP. Furthermore, combining it with a peptide binding to the Ω-loop pocket gives an additive inhibitory effect on the YAP:TEAD interaction. Overall, our results show that it is possible to identify nanomolar inhibitors of the YAP:TEAD interaction that bind to the α-helix pocket, suggesting that developing such compounds might be a strategy to treat cancers where the Hippo pathway is deregulated.

Gene-signature-derived IC50s/EC50s reflect the potency of causative upstream targets and downstream phenotypes.

Multiplexed gene-signature-based phenotypic assays are increasingly used for the identification and profiling of small molecule-tool compounds and drugs. Here we introduce a method (provided as R-package) for the quantification of the dose-response potency of a gene-signature as EC50 and IC50 values. Two signaling pathways were used as models to validate our methods: beta-adrenergic agonistic activity on cAMP generation (dedicated dataset generated for this study) and EGFR inhibitory effect on cancer cell viability. In both cases, potencies derived from multi-gene expression data were highly correlated with orthogonal potencies derived from cAMP and cell growth readouts, and superior to potencies derived from single individual genes. Based on our results we propose gene-signature potencies as a novel valid alternative for the quantitative prioritization, optimization and development of novel drugs.

Helios: History and Anatomy of a Successful In-House Enterprise High-Throughput Screening and Profiling Data Analysis System.

We describe the main characteristics of the Novartis Helios data analysis software system (Novartis, Basel, Switzerland) for plate-based screening and profiling assays, which was designed and built about 11 years ago. It has been in productive use for more than 10 years and is one of the important standard software applications running for a large user community at all Novartis Institutes for BioMedical Research sites globally. A high degree of automation is reached by embedding the data analysis capabilities into a software ecosystem that deals with the management of samples, plates, and result data files, including automated data loading. The application provides a series of analytical procedures, ranging from very simple to advanced, which can easily be assembled by users in very flexible ways. This also includes the automatic derivation of a large set of quality control (QC) characteristics at every step. Any of the raw, intermediate, and final results and QC-relevant quantities can be easily explored through linked visualizations. Links to global assay metadata management, data warehouses, and an electronic lab notebook system are in place. Automated transfer of relevant data to data warehouses and electronic lab notebook systems are also implemented.

Theoretical and experimental relationships between percent inhibition and IC50 data observed in high-throughput screening.

The four-parameter logistic Hill equation models the theoretical relationship between inhibitor concentration and response and is used to derive IC(50) values as a measure of compound potency. This relationship is the basis for screening strategies that first measure percent inhibition at a single, uniform concentration and then determine IC(50) values for compounds above a threshold. In screening practice, however, a "good" correlation between percent inhibition values and IC(50) values is not always observed, and in the literature, there seems confusion about what correlation even to expect. We examined the relationship between percent inhibition data and IC(50) data in HDAC4 and ENPP2 high-throughput screening (HTS) data sets and compared our findings with a series of numerical simulations that allowed the investigation of the influence of parameters representing different types of uncertainties: variability in the screening concentration (related to solution library and compound characteristics, liquid handling), variations in Hill model parameters (related to interaction of compounds with target, type of assay), and influences of assay data quality parameters (related to assay and experimental design, liquid handling). In the different sensitivity analyses, we found that the typical variations of the actual compound concentrations in existing screening libraries generate the largest contributions to imperfect correlations. Excess variability in the ENPP2 assay above the values of the simulation model can be explained by compound aggregation artifacts.

Comparison of multivariate data analysis strategies for high-content screening.

High-content screening (HCS) is increasingly used in biomedical research generating multivariate, single-cell data sets. Before scoring a treatment, the complex data sets are processed (e.g., normalized, reduced to a lower dimensionality) to help extract valuable information. However, there has been no published comparison of the performance of these methods. This study comparatively evaluates unbiased approaches to reduce dimensionality as well as to summarize cell populations. To evaluate these different data-processing strategies, the prediction accuracies and the Z' factors of control compounds of a HCS cell cycle data set were monitored. As expected, dimension reduction led to a lower degree of discrimination between control samples. A high degree of classification accuracy was achieved when the cell population was summarized on well level using percentile values. As a conclusion, the generic data analysis pipeline described here enables a systematic review of alternative strategies to analyze multiparametric results from biological systems.

Compound set enrichment: a novel approach to analysis of primary HTS data.

The main goal of high-throughput screening (HTS) is to identify active chemical series rather than just individual active compounds. In light of this goal, a new method (called compound set enrichment) to identify active chemical series from primary screening data is proposed. The method employs the scaffold tree compound classification in conjunction with the Kolmogorov-Smirnov statistic to assess the overall activity of a compound scaffold. The application of this method to seven PubChem data sets (containing between 9389 and 263679 molecules) is presented, and the ability of this method to identify compound classes with only weakly active compounds (potentially latent hits) is demonstrated. The analysis presented here shows how methods based on an activity cutoff can distort activity information, leading to the incorrect activity assignment of compound series. These results suggest that this method might have utility in the rational selection of active classes of compounds (and not just individual active compounds) for followup and validation.

Integration of multiple readouts into the z' factor for assay quality assessment.

Methods that monitor the quality of a biological assay (i.e., its ability to discriminate between positive and negative controls) are essential for the development of robust assays. In screening, the most commonly used parameter for monitoring assay quality is the Z' factor, which is based on 1 selected readout. However, biological assays are able to monitor multiple readouts. For example, novel multiparametric screening technologies such as high-content screening provide information-rich data sets with multiple readouts on a compound's effect. Still, assay quality is commonly assessed by the Z' factor based on a single selected readout. This report suggests an extension of the Z' factor, which integrates multiple readouts for assay quality assessment. Using linear projections, multiple readouts are condensed to a single parameter, based on which the assay quality is monitored. The authors illustrate and evaluate this approach using simulated data and real-world data from a high-content screen. The suggested approach is applicable during assay development, to optimize the image analysis, as well as during screening to monitor assay robustness. Furthermore, data sets from high-content imaging assays and other state-of-the-art multiparametric screening technologies, such as flow cytometry or transcript analysis, could be analyzed.

Assay data quality assessment.

An overview of the characteristics of classical and outlier-resistant data summaries is provided. The latter are important because outlier data can skew results and decisions based on them. The simple data summaries are the basis for all composite assay and screening data quality measures, for example, the signal-to-noise ratio, signal-to-background ratio, assay and screening window coefficients Z ' and Z, or strictly standardized mean difference (SSMD). In addition to the measures of assay reliability which are based on assessing the size of the "signal windows," some measures for the characterization of the degree of agreement of repeated measurements are also outlined.

Comparison of variability and sensitivity between nuclear translocation and luciferase reporter gene assays.

High-content screening (HCS), a technology based on subcellular imaging by automated microscopy and sophisticated image analysis, has emerged as an important platform in small-molecule screening for early drug discovery. To validate a subcellular imaging assay for primary screening campaigns, an HCS assay was compared with a non-image-based readout in terms of variability and sensitivity. A study was performed monitoring the accumulation of the forkhead transcription factor of the O subfamily (FOXO3a) coupled with green fluorescent protein in the nucleus of human osteosarcoma (U-2 OS) cells. In addition, the transcription of a luciferase gene coupled with a FOXO3a-responsive promoter was monitored. This report demonstrates that both assay formats show good reproducibility in primary and concentration response screening despite differences in statistical assay quality. In primary screening, the correlation of compound activity between the 2 assays was low, in contrast to the good correlation of the IC(50) values of confirmed compounds. Furthermore, the high-content imaging assay showed a mean shift of 2.63-fold in IC(50) values compared with the reporter gene assay. No chemical scaffold was specifically found with 1 of the technologies only, however these results validate the HCS technology against established assays for screening of new molecular entities.

Recommendations for the reduction of compound artifacts in time-resolved fluorescence resonance energy transfer assays.

Time-resolved (TR) fluorescence resonance energy transfer (FRET) is a widely accepted technology for high throughput screening (HTS), being able to detect and quantify the interactions of specific biomolecules in a homogeneous format. TR-FRET has several advantages for HTS applications that reduce assay artifacts such as compound interference. However, in some cases artifacts due to compound autofluorescence, color quenching, or signal stability are still observed. This report presents strategies addressing these issues by several means. One recommendation is the recording and visualization of differences in the donor/acceptor fluorescence, which allows the identification of compound artifacts. Another suggestion is to adjust the time delay, between excitation and recording of the fluorescence, in order to reduce compound interference. Furthermore, configuring the assay to allow the TR-FRET measurement to be taken at different time points, creating a reaction time course, allows background correction for each sample. Finally, the optimization of the FRET pair, to ensure assay signal stability under screening conditions, can improve the assay quality. This report presents examples of how these simple steps can be applied to enhance the quality of TR-FRET screening campaigns.

A multiparameter screening assay to assess the cytokine-induced expression of endothelial cell adhesion molecules.

Compounds which inhibit endothelial cell inflammatory responses are believed to be of therapeutic value. The cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 play important roles in inflammatory reactions by mediating leukocyte-endothelial interactions. To identify compounds which inhibit the expression of these adhesion molecules following cytokine stimulation we developed an assay which measures E-selectin, ICAM-1, and VCAM-1 in the same experiment. For this, we have taken advantage of the technology of time-resolved fluorimetry, which allows detection of several parameters in parallel, employing anti-E-selectin antibody labeled with europium, and anti-ICAM-1 and anti-VCAM-1 labeled with samarium and terbium, respectively. These antibodies were used to detect the respective antigens in human endothelial cells stimulated with TNFalpha or IL-1beta. In cross-competition assays these antibodies were found to bind specifically to TNF- or IL-1-stimulated cells. This assay, in which three parameters are measured in the same experiment, proved to be robust with signal to noise ratios of 25-35 for E-Selectin, 4-8 for ICAM-1, and 3-9 for VCAM-1. The assay proved to be reproducible in high-throughput screening. The experience with this assay demonstrates that multiple parameters can be measured in an enzyme-linked immunosorbent assay-type assay on cells by using time-resolved fluorimetry. The possibility of obtaining several parameters from one experiment is feasible under high-throughput screening conditions and is of interest for other experimental setups in which the simultaneous measurement of several parameters is desired.