Human induced pluripotent stem cell-derived planar neural organoids assembled on synthetic hydrogels.

The tailorable properties of synthetic polyethylene glycol (PEG) hydrogels make them an attractive substrate for human organoid assembly. Here, we formed human neural organoids from iPSC-derived progenitor cells in two distinct formats: (i) cells seeded on a Matrigel surface; and (ii) cells seeded on a synthetic PEG hydrogel surface. Tissue assembly on synthetic PEG hydrogels resulted in three dimensional (3D) planar neural organoids with greater neuronal diversity, greater expression of neurovascular and neuroinflammatory genes, and reduced variability when compared with tissues assembled upon Matrigel. Further, our 3D human tissue assembly approach occurred in an open cell culture format and created a tissue that was sufficiently translucent to allow for continuous imaging. Planar neural organoids formed on PEG hydrogels also showed higher expression of neural, vascular, and neuroinflammatory genes when compared to traditional brain organoids grown in Matrigel suspensions. Further, planar neural organoids contained functional microglia that responded to pro-inflammatory stimuli, and were responsive to anti-inflammatory drugs. These results demonstrate that the PEG hydrogel neural organoids can be used as a physiologically relevant in vitro model of neuro-inflammation.

Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion.

The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors, and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promoted endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels show superior sensitivity and reproducibility over Matrigel when evaluating known inhibitors, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the US Environmental Protection Agency. The identified synthetic hydrogels should be suitable alternatives to Matrigel for common cell-culture applications.

High-Content Assay Multiplexing for Vascular Toxicity Screening in Induced Pluripotent Stem Cell-Derived Endothelial Cells and Human Umbilical Vein Endothelial Cells.

Endothelial cells (ECs) play a major role in blood vessel formation and function. While there is longstanding evidence for the potential of chemical exposures to adversely affect EC function and vascular development, the hazard potential of chemicals with respect to vascular effects is not routinely evaluated in safety assessments. Induced pluripotent stem cell (iPSC)-derived ECs promise to provide a physiologically relevant, organotypic culture model that is amenable for high-throughput (HT) EC toxicant screening and may represent a viable alternative to traditional in vitro models, including human umbilical vein endothelial cells (HUVECs). To evaluate the utility of iPSC-ECs for multidimensional HT toxicity profiling of chemicals, both iPSC-ECs and HUVECs were exposed to selected positive (angiogenesis inhibitors, cytotoxic agents) and negative compounds in concentration response for either 16 or 24 h in a 384-well plate format. Furthermore, chemical effects on vascularization were quantified using EC angiogenesis on biological (Geltrex™) and synthetic (SP-105 angiogenesis hydrogel) extracellular matrices. Cellular toxicity was assessed using high-content live cell imaging and the CellTiter-Glo® assay. Assay performance indicated good to excellent assay sensitivity and reproducibility for both cell types investigated. Both iPSC-derived ECs and HUVECs formed tube-like structures on Geltrex™ and hydrogel, an effect that was inhibited by angiogenesis inhibitors and cytotoxic agents in a concentration-dependent manner. The quality of HT assays in HUVECs was generally higher than that in iPSC-ECs. Altogether, this study demonstrates the capability of ECs for comprehensive assessment of the biological effects of chemicals on vasculature in a HT compatible format.

LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status.

Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD) (Farrer et al., 2005; Skipper et al., 2005; Di Fonzo et al., 2006; Healy et al., 2008; Paisan-Ruiz et al., 2008; Lesage et al., 2010). LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955, and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955, and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro, and Tyr1699Cys, can positively enhance LRRK2 kinase activity, while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and Okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

Detection of allosteric kinase inhibitors by displacement of active site probes.

Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

Transformation of in vitro tools for kinase profiling: keeping an eye over the off-target liabilities.

INTRODUCTION: Over the past decade, there has been an increased number of FDA approved small molecule kinase inhibitors for the treatment of cancer. This is due, in part, to an increased understanding of the fundamental aspects of kinase biology, coupled with advances in the methods used to study the inhibitory effects of small molecules on kinase activity. Underlying the development of these inhibitors are profiling methods that are used to assess the effect of potential compounds against their desired and undesired targets. The advancement of kinase profiling has stemmed from the development of basic assay technology that allows compounds to be tested against ever larger panels of kinases in a robust, cost-effective manner. Methods have also been developed that rapidly assess compound activity against specific activation states of kinases. There has also been a development of newer methods that move beyond traditional biochemical formats, which take a 'whole cell' approach to compound profiling. AREAS COVERED: This review provides an overview of traditional biochemical-based kinase profiling as well as an introduction to advances that have been made by moving compound profiling into a cell-based format. EXPERT OPINION: While central to the appropriate prioritization and optimization of compounds during the hit to lead phase of early-stage pharmaceutical development, every compound profiling format must be critically assessed so that one can make informed decisions through an understanding of their strengths and limitations. These decisions will ultimately be balanced against cost, complexity and its biological relevance.

Multi-pathway cellular analysis of compound selectivity.

Signaling pathways and their protein target constituents (e.g. kinases) have become important therapeutic targets in many disease areas. Traditional selectivity profiling for kinase inhibitors has relied upon screening panels of recombinant enzymes in biochemical assay formats. Recent studies have highlighted the importance of using cellular assays to better approximate true biological selectivity. We have developed a portfolio of CellSensor beta-lactamase transcriptional reporter gene assays that can be used to screen for perturbagens of various endogenous signaling pathways. Here we describe a multi-pathway profiling approach for generating compound-pathway selectivity maps. To demonstrate the utility of this approach, we have screened 32 known compounds across a diverse panel of 12 key signaling pathways and generated the first comprehensive cellular pathway selectivity profiles of several clinically approved kinase and other well-known bioactive inhibitors. Selectivity score comparisons identified several kinase inhibitors that were more promiscuous than predicted by traditional biochemical profiling methods. For example, we identified effects of sorafenib on the JAK/STAT pathway and demonstrated the potential therapeutic indication of sorafenib in treating leukemia/myeloproliferative disorder patients harboring TEL-JAK2 or JAK2V617F mutations. Our results indicate that multi-pathway profiling can efficiently characterize both on and off-pathway compound activities, revealing potential novel pathways and opportunities for drug repositioning purposes and/or safety liabilities in one profiling campaign.

Development and applications of a broad-coverage, TR-FRET-based kinase binding assay platform.

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor 647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

Sarcoglycan complex: implications for metabolic defects in muscular dystrophies.

The sarcoglycans are known as an integral subcomplex of the dystrophin glycoprotein complex, the function of which is best characterized in skeletal muscle in relation to muscular dystrophies. Here we demonstrate that the white adipocytes, which share a common precursor with the myocytes, express a cell-specific sarcoglycan complex containing beta-, delta-, and epsilon-sarcoglycan. In addition, the adipose sarcoglycan complex associates with sarcospan and laminin binding dystroglycan. Using multiple sarcoglycan null mouse models, we show that loss of alpha-sarcoglycan has no consequence on the expression of the adipocyte sarcoglycan complex. However, loss of beta- or delta-sarcoglycan leads to a concomitant loss of the sarcoglycan complex as well as sarcospan and a dramatic reduction in dystroglycan in adipocytes. We further demonstrate that beta-sarcoglycan null mice, which lack the sarcoglycan complex in adipose tissue and skeletal muscle, are glucose-intolerant and exhibit whole body insulin resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscles. Thus, our data demonstrate a novel function of the sarcoglycan complex in whole body glucose homeostasis and skeletal muscle metabolism, suggesting that the impairment of the skeletal muscle metabolism influences the pathogenesis of muscular dystrophy.

Pharmacological characterization of purified recombinant mTOR FRB-kinase domain using fluorescence-based assays.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in nutrient sensing and cell growth and is a validated target for oncology and immunosuppression. Two modes of direct small-molecule inhibition of mTOR activity are known: targeting of the kinase active site and a unique mode in which the small molecule rapamycin, in complex with FKBP12 (the 12-kDa FK506 binding protein), binds to the FRB (FKBP12/rapamycin binding) domain of mTOR and inhibits kinase activity through a poorly defined mechanism. To facilitate the study of these processes, the authors have expressed and purified a truncated version of mTOR that contains the FRB and kinase domains and have developed homogeneous fluorescence-based assays to study mTOR activity. They demonstrate the utility of these assays in studies of active site-directed and FRB domain-directed mTOR inhibition. The results suggest that these assays can replace traditional radiometric or Western blot-based assays.

A fluorescence lifetime based binding assay to characterize kinase inhibitors.

The authors present a fluorescence lifetime-based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)-binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states.

An improved beta-lactamase reporter assay: multiplexing with a cytotoxicity readout for enhanced accuracy of hit identification.

A problem inherent to the use of cellular assays for drug discovery is their sensitivity to cytotoxic compounds, which can result in false hits from certain compound screens. To alleviate the need to follow-up hits from a reporter assay with a separate cytotoxicity assay, the authors have developed a multiplexed assay that combines the readout of a beta-lactamase reporter with that of a homogeneous cytotoxicity indicator. Important aspects to the development of the multiplexed format are addressed, including results that demonstrate that the IC(50) values of 40 select compounds in a beta-lactamase reporter assay for nuclear factor kappa B and SIE pathway antagonists are not affected by the addition of the cytotoxicity indicator. To demonstrate the improvement in hit confirmation, the multiplexed assay was used to perform a small-library screen (7728 compounds) for serotonin 5HT1A receptor antagonists. Hits identified from analysis of the beta-lactamase reporter data alone were compared to those hits determined when the reporter and cytotoxicity data generated from the multiplexed assay were combined. Confirmation rates were determined from compound follow-up using dose-response analysis of the potential antagonist hits identified by the initial screen. In this representative screen, the multiplexed assay approach yielded a 19% reduction in the number of compounds flagged for follow-up, with a 37% decrease in the number of false hits, demonstrating that multiplexing a beta-lactamase reporter assay with a cytotoxicity readout is a highly effective strategy for reducing false hit rates in cell-based compound screening assays.

Analysis of ligand-dependent recruitment of coactivator peptides to estrogen receptor using fluorescence polarization.

Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ERalpha or ERbeta in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ERalpha and ERbeta with affinity ranging from 20-500 nm. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17beta-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC50) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17beta-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC50 values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERbeta was observed in the presence of the ERbeta-selective agonist diarylpropionitrile, but not the ERalpha-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.

High-throughput screening assays for CYP2B6 metabolism and inhibition using fluorogenic vivid substrates.

CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo- and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme's involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K(i), K(d), or K(m) values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

A high throughput screening assay to screen for CYP2E1 metabolism and inhibition using a fluorogenic vivid p450 substrate.

Large-scale screening of multiple compound libraries and combinatorial libraries for pharmacological activity is one of the novel approaches of the modern drug discovery process. The application of isozyme-specific high-throughput screening (HTS) assays for characterizing the interactions of potential drug candidates with major human drug-metabolizing cytochrome p450 enzymes (p450s) is newly becoming an essential part of this process. Fluorescence-based HTS assays have been successfully employed for in vitro assessment of drug-drug interactions and enzyme inhibition with several p450 isoforms, including CYP3A4, CYP2D6, CYP2C9, and CYP2C19. Here we describe a fluorescence-based HTS assay for detecting drug metabolism and inhibition with human CYP2E1. CYP2E1 plays an important role in the metabolism of several drugs, many solvents, and toxins and therefore has been repeatedly linked to numerous pathologies, including cancer, liver and kidney toxicity, diabetes, and alcoholism. The assay is based on the ability of a drug to compete with the fluorogenic Vivid CYP2E1 Blue Substrate for CYP2E1 metabolism and thus enables rapid screening of lead molecules for their inhibitory potential. We have used this assay to screen a panel of drugs and compounds for their effects on CYP2E1 metabolism and inhibition. Our results demonstrate the assay's usefulness in identifying CYP2E1 substrates and inhibitors and in enabling in-depth characterization of their interactions with the CYP2E1 isozyme. We also present detailed characteristics of the assay, including its dynamic range and Z'-factor values, which indicate that this robust assay is well suited for kinetic and inhibition studies in HTS formats.