A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries.

CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a ß-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z' score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z' ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.

Determining direct binders of the Androgen Receptor using a high-throughput Cellular Thermal Shift Assay.

Androgen Receptor (AR) is a key driver in prostate cancer. Direct targeting of AR has valuable therapeutic potential. However, the lack of disease relevant cellular methodologies capable of discriminating between inhibitors that directly bind AR and those that instead act on AR co-regulators has made identification of novel antagonists challenging. The Cellular Thermal Shift Assay (CETSA) is a technology enabling confirmation of direct target engagement with label-free, endogenous protein in living cells. We report the development of the first high-throughput CETSA assay (CETSA HT) to identify direct AR binders in a prostate cancer cell line endogenously expressing AR. Using this approach, we screened a pharmacology library containing both compounds reported to directly engage AR, and compounds expected to target AR co-regulators. Our results show that CETSA HT exclusively identifies direct AR binders, differentiating them from co-regulator inhibitors where other cellular assays measuring functional responses cannot. Using this CETSA HT approach we can derive apparent binding affinities for a range of AR antagonists, which represent an intracellular measure of antagonist-receptor Ki performed for the first time in a label-free, disease-relevant context. These results highlight the potential of CETSA HT to improve the success rates for novel therapeutic interventions directly targeting AR.

Assessment of the Target Engagement and D-Serine Biomarker Profiles of the D-Amino Acid Oxidase Inhibitors Sodium Benzoate and PGM030756.

Irregular N-methyl-D-aspartate receptor (NMDAR) function is one of the main hypotheses employed to facilitate understanding of the underlying disease state of schizophrenia. Although direct agonism of the NMDAR has not yielded promising therapeutics, advances have been made by modulating the NMDAR co-agonist site which is activated by glycine and D-serine. One approach to activate the co-agonist site is to increase synaptic D-serine levels through inhibition of D-amino acid oxidase (DAO), the major catabolic clearance pathway for this and other D-amino acids. A number of DAO inhibitors have been developed but most have not entered clinical trials. One exception to this is sodium benzoate which has demonstrated efficacy in small trials of schizophrenia and Alzheimer's disease. Herein we provide data on the effect of sodium benzoate and an optimised Takeda compound, PGM030756 on ex vivo DAO enzyme occupancy and cerebellar D-serine levels in mice. Both compounds achieve high levels of enzyme occupancy; although lower doses of PGM030756 (1, 3 and 10 mg/kg) were required to achieve this compared to sodium benzoate (300, 1000 mg/kg). Cerebellar D-serine levels were increased by both agents with a delay of approximately 6 h after dosing before the peak effect was achieved. Our data and methods may be useful in understanding the effects of sodium benzoate that have been seen in clinical trials of schizophrenia and Alzheimer's disease and to support the potential clinical assessment of other DAO inhibitors, such as PGM030756, which demonstrate good enzyme occupancy and D-serine increases following administration of low oral doses.

Anti-hyperalgesic effects of a novel TRPM8 agonist in neuropathic rats: a comparison with topical menthol.

Menthol has historically been used topically to alleviate various pain conditions. At low concentrations, this non-selective TRPM8 agonist elicits a cooling sensation, however higher concentrations result in cold hyperalgesia in normal subjects and paradoxically analgesia in neuropathic patients. Through behavioural and electrophysiological means, we examined whether this back-translated into a pre-clinical rodent model. Menthol was applied topically to the hind paws of naive and spinal nerve-ligated (SNL) rats. In behavioural assays, menthol did not affect withdrawal thresholds to mechanical stimulation and 10% and 40% menthol rarely sensitised withdrawals to innocuous cooling in naïve rats. However, in SNL rats, 10% and 40% menthol alleviated cold hypersensitivity. This was partly corroborated by in vivo electrophysiological recordings of dorsal horn lamina V/VI neurones. As several studies have implicated TRPM8 in analgesia, we examined whether a novel systemically available TRPM8 agonist, M8-Ag, had more potent anti-hyperalgesic effects than menthol in neuropathic rats. In vitro, M8-Ag activates TRPM8, expressed in HEK293 cells, with an EC50 of 44.97 nM. In vivo, M8-Ag inhibited neuronal responses to innocuous and noxious cooling in SNL rats with no effect in sham-operated rats. This effect was modality selective; M8-Ag did not alter neuronal responses to mechanical, heat or brush stimulation. In addition, M8-Ag attenuated behavioural hypersensitivity to innocuous cooling but not mechanical stimulation. These data suggest that menthol induced hyperalgesia is not consistently replicable in the rat and that the analgesic properties are revealed by injury. Systemic TRPM8 agonists might be beneficial in neuropathy without affecting normal cold sensitivity.

Identification of a novel GPR81-selective agonist that suppresses lipolysis in mice without cutaneous flushing.

GPR81, which exhibits a high degree of homology with GPR109a, has been recently identified as a lactate receptor. Similar to GPR109a, the activation of GPR81 by lactate suppresses lipolysis, suggesting that GPR81 may be a potential drug target for treating dyslipidemia. In addition, the fact that GPR81 is expressed only in adipocytes, whereas GPR109a is expressed in various tissues and cells, including Langerhans cells, which are considered responsible for flushing, indicates that targeting GPR81 could lead to the development of antidyslipidemia agents with a reduced risk of this side effect. However, the pharmacological role of GPR81 remains largely unclear, mainly because of the lack of potent and selective surrogate GPR81 agonists suitable for in vivo studies. In the present study, we showed that lactate-induced suppression of lipolysis in explants of white adipose tissue (WAT) depends on the presence of GPR81. We also performed high-throughput screening (HTS) and identified four novel chemical clusters as GPR81 agonists. Chemical optimization of aminothiazole derivatives led to the discovery of a lead compound with improved potency. The compound inhibited lipolysis in differentiated 3T3-L1 adipocytes. Finally, intraperitoneal administration of this compound suppressed lipolysis in mice at doses that did not cause cutaneous flushing. This is the first description of a 50nM GPR81 selective agonist with in vivo efficacy, without the side effect, i.e., flushing. These results suggest that GPR81 is an attractive drug target for treating dyslipidemia without the risk of flushing.

Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays.

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

A morphology- and kinetics-based cascade for human neural cell high content screening.

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits represents a novel mechanism for treating a variety of central nervous system disorders. Using human neural precursor cultures and a variety of assays for studying stem cell behavior we have screened two libraries of commercially available compounds using an endpoint high content screening assay. We then performed detailed follow-up mechanistic studies on confirmed hits using endpoint and kinetics assays to characterize and differentiate the mechanisms of action of these compounds. The screening cascade employed successfully identified a number of active compounds with differing mechanisms of action. This approach shows how hits from a phenotypic screen can be prioritized and characterized by high content screening to identify potentially novel mechanisms and druggable targets to take forward into more conventional high-throughput screening approaches.

Heteromerization and colocalization of TrpV1 and TrpV2 in mammalian cell lines and rat dorsal root ganglia.

Coassociation of the vanilloid transient receptor potential (Trp) ion channels, TrpV1 and TrpV2, was investigated by immunoprecipitation and immunofluorescence in transfected mammalian cell lines, rat dorsal root ganglia and spinal cord. TrpV1/TrpV2 heteromeric complexes were coimmunoprecipitated from human embryonic kidney cells and F-11 dorsal root ganglion hybridoma cells following their transient coexpression. Immunofluorescent labelling of transfected F-11 cells revealed colocalization of TrpV1 and TrpV2 at the cell surface. Immunoprecipitation from rat dorsal root ganglion lysates identified a minor population of receptor complexes composed of TrpV1/TrpV2 heteromers, consistent with a small proportion of cells double-labelled with TrpV1 and TrpV2 antibodies in rat dorsal root ganglion sections. TrpV1/TrpV2 receptor complexes may represent a functionally distinct ion channel complex that may increase the diversity observed within the Trp ion channel family.

Quantitative assays of chemotaxis and chemokinesis for human neural cells.

Cell migration is vital for many physiological processes, and its modulation is likely to be of therapeutic benefit. In this study we have developed fluorescence image-based chemokinesis and chemotaxis assays on the Cellomics (Pittsburgh, PA) ArrayScan platform, which would be suitable for industrial drug discovery in a variety of fields. Studying the migratory characteristics of neural stem cells is of interest for understanding the therapeutic potential of these cells, in terms of both cellular transplantation and the activation of endogenous populations of stem cells. Growth conditions were identified whereby human neural precursors could be maintained as neurospheres and plated out into microtitre plates for high-content assays. Chemokinesis was assessed using fluorescent bead-coated 96-well microtitre plates, whilst chemotaxis was assessed using BD Biosciences (Oxford, UK) Fluoroblok 24-well plates. Assays for both chemokinesis and chemotaxis were developed that were quantified automatically using the ArrayScan and appropriate algorithms. Using the two complementary techniques, foetal bovine serum was observed to have chemokinetic effects on the cells, whilst platelet-derived growth factor isoform AB was chemotactic. The two assays described here are suitable for screening for novel modulators of cell migration, or for performing more detailed mechanistic follow-up studies. These assays enable us to perform cell motility studies with minimal laboratory handling, in an automated manner, thereby allowing quantitative studies of cell behaviour to be incorporated in a routine drug discovery screening cascade.