Christian N. Parker

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5.

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4−/− crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.

Inhibition profiling of human carbonic anhydrase II by high-throughput screening of structurally diverse, biologically active compounds.

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of ∼2%. Potential hits were further characterized by determining their IC50 and Kd values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.

YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors

The Yes‐associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down‐regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug‐induced apoptosis. We further show that the proangiogenic microfibrillar‐associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31+ vasculature. Conclusions: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins. (Hepatology 2015;62:1497–1510)

A Screen for Enhancers of Clearance Identifies Huntingtin as a Heat Shock Protein 90 (Hsp90) Client Protein

Background: Molecular chaperones assist in the folding of metastable proteins implicated in neurodegenerative diseases. Results: Huntingtin is a heat shock protein 90 (Hsp90) client protein. Conclusion: Hsp90 inhibition-mediated degradation of soluble mutant huntingtin is independent of a cellular heat shock response. Significance: Mechanisms targeting Hsp90 chaperone function may provide new treatments for Huntington disease. Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.

Identification of the C3a Receptor (C3AR1) as the Target of the VGF-derived Peptide TLQP-21 in Rodent Cells

Background: TLQP-21 is a bioactive peptide for which the receptor(s) are unknown. Results: We demonstrate that C3AR1 is a receptor for TLQP-21. Conclusion: Many of the effects of TLQP-21 can be explained by C3AR1 activation. Significance: These results provide a bridge linking the regulation of metabolism and the activation of complement in rodents. TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

“Plate Cherry Picking”: A Novel Semi-Sequential Screening Paradigm for Cheaper, Faster, Information-Rich Compound Selection

This work describes a novel semi-sequential technique for in silico enhancement of high-throughput screening (HTS) experiments now employed at Novartis. It is used in situations in which the size of the screen is limited by the readout (e.g., high-content screens) or the amount of reagents or tools (proteins or cells) available. By performing computational chemical diversity selection on a per plate basis (instead of a per compound basis), 25% of the 1,000,000-compound screening was optimized for general initial HTS. Statistical models are then generated from target-specific primary results (percentage inhibition data) to drive the cherry picking and testing from the entire collection. Using retrospective analysis of 11 HTS campaigns, the authors show that this method would have captured on average two thirds of the active compounds (IC50 < 10 µM) and three fourths of the active Murcko scaffolds while decreasing screening expenditure by nearly 75%. This result is true for a wide variety of targets, including G-protein-coupled receptors, chemokine receptors, kinases, metalloproteinases, pathway screens, and protein-protein interactions. Unlike time-consuming “classic” sequential approaches that require multiple iterations of cherry picking, testing, and building statistical models, here individual compounds are cherry picked just once, based directly on primary screening data. Strikingly, the authors demonstrate that models built from primary data are as robust as models built from IC50 data. This is true for all HTS campaigns analyzed, which represent a wide variety of target classes and assay types. (Journal of Biomolecular Screening 2007:320-327)

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.

A review of methods to monitor the modulation of mRNA stability: a novel approach to drug discovery and therapeutic intervention.

Posttranscriptional regulation of gene expression is an elaborate and intricate process, constituting an important mechanism for the control of protein expression. During its existence, mRNA is escorted by proteins and other RNAs, which control the maturation, transportation, localization, translational efficiency, and ultimately its degradation. Without changes at the transcription level, mRNA steady-state levels can vary dramatically by just small changes in mRNA stability. By influencing the metabolism of specific mRNAs, the abundance of specific mRNAs can be controlled in organisms from bacteria to mammals. In eukaryotic cells, the control of mRNA stability is exerted through specific cis-acting elements (sequence-specific control elements) and trans-acting factors (mRNA binding proteins and some miRNAs). mRNA stability appears to be a key regulator in controlling the expression of many proteins. Dysregulation of mRNA stability has been associated with human diseases, including cancer, inflammatory disease, and Alzheimer’s. These observations suggest that modulating the stability of specific mRNAs may represent a viable strategy for pharmaceutical intervention. The literature already describes several compounds that influence mRNA stability. Measuring mRNA stability by conventional methods is labor intensive and time-consuming. However, several systems have been described that can be used to screen for modulators of mRNA levels in a high-throughput format. Thus, these assay systems offer a novel approach for screening targets that at present appear to be poorly “drugable.” This review describes the utility of mRNA stability as a novel approach to drug discovery, focusing on assay methods and tool compounds available to monitor mRNA stability. The authors describe mRNA stability assays and issues related to this approach.

Development of a Method for the High-Throughput Quantification of Cellular Proteins

Hunting for huntingtin: We describe a screening assay based on the inducible expression of the mutant huntingtin protein in cells and on its highly sensitive homogenous determination. Rapid, reproducible, and robust protein determination was achieved through the use of two donor–acceptor‐labeled antibodies and time‐resolved FRET. The assay was developed and validated for ultra‐throughput screening of low‐molecular‐weight compounds modulating the expression of the mutant protein.

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.