High throughput (HT) screening is at the starting point for most drug discovery pro- grams. As the range of targets being pursued widens new technologies have to be deployed to enable assays built to measure the activity of proteins previously deemed challenging. Flow cytometry is a technology providing multi-parametric analysis of single cells or other particles in suspension, such as beads. High throughput (HT) flow cytometry has become a very attractive screening platform for drug discovery. In this chapter we describe a 1536 well format high throughput screen of 500,000 compounds to find inhibitors of Rac1 GTPase to prevent allergic airway hyper-responsiveness in asthma. We discuss the assay development, miniaturization and validation carried out prior to the full screening campaign. We then describe how we have automated our iQue ® HD screener instruments and how we proceed with the data analysis and explain why we chose to run this screen on a flow cytometer and how it enabled us to reduce cost and timelines for the project.

Ras and Ras-related small GTPases are key regulators of diverse cellular functions that impact cell growth, survival, motility, morphogenesis, and differentiation. They are important targets for studies of disease mechanisms as well as drug discovery. Here, we report the characterization of small molecule agonists of one or more of six Rho, Rab, and Ras family GTPases that were first identified through flow cytometry-based, multiplexed high-throughput screening of 200000 compounds. The activators were categorized into three distinct chemical families that are represented by three lead compounds having the highest activity. Virtual screening predicted additional compounds with potential GTPase activating properties. Secondary dose-response assays performed on compounds identified through these screens confirmed agonist activity of 43 compounds. While the lead and second most active small molecules acted as pan activators of multiple GTPase subfamilies, others showed partial selectivity for Ras and Rab proteins. The compounds did not stimulate nucleotide exchange by guanine nucleotide exchange factors and did not protect against GAP-stimulated GTP hydrolysis. The activating properties were caused by a reversible stabilization of the GTP-bound state and prolonged effector protein interactions. Notably, these compounds were active both in vitro and in cell-based assays, and small molecule-mediated changes in Rho GTPase activities were directly coupled to measurable changes in cytoskeletal rearrangements that dictate cell morphology.

Successful Expression of Adenovirus E3 CR1 Genes from constructs carrying Adenovirus Tripartite Leader sequences: a technical breakthrough Camden Bair, Poornima Kotha, and Adriana Kajon Over 70 human adenovirus genotypes have been described to date and classified into seven species (HAdV-A to -G). One of the few characteristics distinguishing HAdV genomes of different species is the unique repertoire of E3 open reading frames (ORFs) encoding non-structural membrane proteins of unknown function. This species-specific feature likely contributes to the distinct pathogenic traits of each of these groups but the lack of functional information remains a major gap in the field. In our laboratory the characterization of species HAdV-Band HAdV-E-specific E3 ORFs has been hampered by the intriguing difficulty to ectopically express the encoded proteins at adequate levels using traditional mammalian expression vectors. In infected cells transcription of these particular E3 genes is driven by the major late promoter at late time points post infection. All late transcripts carry a 5’ 200 nucleotide untranslated sequence called the tripartite leader (TPL). TPL has been shown to facilitate efficient translation of late transcripts. We cloned small epitope-tagged versions of our ORFs of interest into commercially available vector pCI-Neo (Promega) with no detectable protein expression by Western Blot in lysates of transfected 293T cells. Literature searches brought to our attention vector pMT2 developed by Kaufman et al. that encodes HAdV-C TPL. To our surprise, expression of all 4 tagged proteins from the counterpart pMT2 constructs was readily detectable. We subsequently inserted the HAdV-E TPL sequence upstream of the E3 genes in the original pCI-Neo constructs and rescued protein expression for all cloned ORFs. RT-PCR analysis of gene expression showed that the mRNA levels for our transcripts of interest were also higher in the presence of TPL sequences suggesting that TPL may be contributing to both mRNA stabilization and efficient protein translation. These findings merit further investigation to identify how TPL sequences enhance protein expression of these particular E3 genes. Optogenetic based regulation of autophagy against tauopathies Binder J, Deretic V, Weick J, and Bhaskar K Filamentous aggregation of microtubule associated protein tau, as neurofibrillary tangles (NFTs), is a major pathological hallmark of tauopathies including Alzheimer’s disease (AD). This pathological tau (p-Tau) impairs microtubule function, axonal transport and synaptic function, which in turn strongly correlate with cognitive decline. Multiple reports have suggested impaired autophagic clearance of p-Tau within neurons leads to NFT pathology and neurodegeneration. Transcription factor EB (TFEB), which is a master regulator of autophagy flux, has recently been shown to clear p-Tau, prevent synaptic and behavioral defects in a mouse model of tauopathy. Furthermore, our group has recently demonstrated that induction of autophagy either via FDA approved drugs (Bromhexine/Flubendazole) or by overexpressed TFEB can clear inflammation-induced p-Tau in neuronal cells. However, sustained activation of TFEB and autophagy may pose the risk of burdening cellular bioenergetics and be deleterious during conditions such as ischemic stroke, which is more prevalent in aged individuals. Here we have tested a light-based gene expression system with a goal to achieve precise spatiotemporal control over TFEB expression and thus regulatable autophagy flux in neuronal cells. This technology utilizes an engineered version of EL222, a bacterial transcription factor that contains a Light-Oxygen-Voltage protein, which binds DNA when illuminated with blue light (465nm). HEK293T cells transiently transfected with EL222 and TFEBFlag plasmids displayed robust TFEB-Flag expression when exposed to 465nm light (~2W/m2) for 9 -12 hours compared to no-light condition. As a first step in testing this approach in neuronal system, we tested inducible pluripotent stem cell derived neurons (iPSNs) from Down’s syndrome patient and observed significantly elevated tau oligomers, tau hyperphosphorylated at AT180 and AT8 sites. In the ongoing studies, we are engineering lenti-viral expression system that encodes EL222 and TFEB-Flag to induce autophagy in primary neurons, iPSNs and in rTg4510 mouse model of tauopathy. Specific and Potent Inhibitors of DNA Ligases as Cancer Therapeutics Rhys C Brooks, Yoshihiro Matsumoto, Mark B. Carter, Larry A. Sklar, and Alan E. Tomkinson Human DNA ligases I, III, and IV have unique functions that preserve genomic integrity by joining Okazaki fragments in DNA replication, and completing DNA damage repair through sealing of singleand double-strand DNA breaks. Dysregulation of these ligases has been correlated with reduced survival in leukemia, breast cancer, and neuroblastoma providing evidence for DNA ligases as therapeutic targets in limiting cancer progression. To elucidate such inhibitors we have optimized and validated a highly sensitive fluorescent-based ligation assay which utilizes an Alexa-488 labeled nicked DNA substrate that, when ligated to an upstream quencher, reduces the fluorescence output at a predictable rate. Our group has subsequently adapted this assay to characterize the kinetic and inhibitory properties of various compounds as they affect the nick repair capacity of these ligases. Recently, the compound SCR7 has been identified by others as a ligase IV specific inhibitor proposed to reduce tumor growth. However, we demonstrate that SCR7 is neither a selective nor potent inhibitor of ligases, suggesting it of little role as an effective adjunct. These data provide insight regarding the need for a sensitive and reliable assay to identify ligase inhibitors for cancer therapies. Moreover we have modified this novel fluorescent-based assay for high-throughput use of FDA approved chemical libraries with the aim of repurposing these compounds for cancer treatment. Quantitating dendritic cell clustering in the lymph node Janie Rae Byrum, Matthew Fricke, Justyna Tafoya, Melanie Moses, Judy L Cannon The efficiency of the T cell search for antigen presented by dendritic cells (DCs) in lymph nodes (LNs) is a determinant of the overall timing of the T cell immune response to infection. While there is suggestion that DCs are clustered in LNs, there has been little quantitative analysis done to precisely analyze DC positioning in LNs. We present the quantitation of murine DC motility, surface area, and volume in the lymph node. We also use computational analysis of 2 photon microscopy images of explanted murine lymph nodes from CD11c-YFP mice to determine the degree of clustered-ness of DCs. Our analysis indicates a degree of DC clustering within the lymph node and that T cells and DCs may share positional information. Previously our lab identified sites in the lymph node visited with greater frequency by T cells than would be expected by random motility. We hypothesize such sites demonstrate that DC clusters may actively attract T cells. Elucidating whether T cell motility around DC clusters is distinctive from T cell motility in areas where DCs are non-clustered will help decipher T cell search strategy and the timing of the adaptive immune response. Precision autophagy mediated by TRIMs governs key innate immunity systems Tomonori Kimura, Seong Won Choi, Michael Mandell, Vojo P Deretic The present paradigms of selective autophagy in mammalian cells cannot fully explain the specificity and selectivity of autophagic degradation. In this paper, we report that a subset of tripartite motif (TRIM) proteins act as specialized receptors for highly specific autophagy (precision autophagy) of key components of the inflammasome and type I interferon response systems. TRIM20 targets the inflammasome components, including NLRP3, NLRP1, and pro–caspase 1, for autophagic degradation, whereas TRIM21 targets IRF3. TRIM20 and TRIM21 directly bind their respective cargo and recruit autophagic machinery to execute degradation. The autophagic function of TRIM20 is affected by mutations associated with familial Mediterranean fever. These findings broaden the concept of TRIMs acting as autophagic receptor regulators executing precision autophagy of specific cytoplasmic targets. In the case of TRIM20 and TRIM21, precision autophagy controls the hub signaling machineries and key factors, inflammasome and type I interferon, directing cardinal innate immunity response systems in humans. Pathological tau induces inflammasome activation and neuroinflammation relevant to Alzheimer’s disease Shanya Jiang, Jessica Binder, Nicole Maphis, Lea Weston, Walter Duran, Crina Floruta, Stephen Jett, Eicke Latz and Kiran Bhaskar Hyperphosphorylation and aggregation of tau protein is a pathological
 hallmark of Alzheimer’s disease (AD) and related tauopathies. Our previous studies demonstrated that activation of microglia leads to accelerated tau pathology and cognitive impairment. However, it remains elusive how the microglial activation occurs and precede tau pathology. Here, we show that pathological Tau (p-Tau) is secreted in either exosomedependent or –independent manner from neuroblastoma (N2a) cells expressing human p-Tau. This led to uptake of p-Tau by BV2 microglial cells, expression of various proinflammatory genes and activation of inflammasome. Incubation of ASC-cerulean macrophages with paired helical filaments (PHFs) purified from rTg4510 transgenic mice brains or from human AD patient brain resulted in inflammasome activation and IL1 secretion into the media. Furthermore, rTg4510 mice showed age-dependent increase in the pro-inflammatory gene expression, which can be mitigated by the suppression of human tau via doxycycline treatment. Taken together, our results suggest that p-Tau derived from neurons can be taken up into the microglia and serve as damageassociated m

Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3'-5'-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing.

A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

Stroke is a leading cause of death and disability and treatment options are limited. A promising approach to accelerate the development of new therapeutics is the use of high-throughput screening of chemical libraries. Using a cell-based high-throughput oxygen-glucose deprivation (OGD) model, we evaluated 1,200 small molecules for repurposed application in stroke therapy. Isoxsuprine hydrochloride was identified as a potent neuroprotective compound in primary neurons exposed to OGD. Isoxsuprine, a β2-adrenergic agonist and NR2B subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonist, demonstrated no loss of efficacy when administered up to an hour after reoxygenation in an in vitro stroke model. In an animal model of transient focal ischemia, isoxsuprine significantly reduced infarct volume compared to vehicle (137±18 mm3 versus 279±25 mm3, p<0.001). Isoxsuprine, a peripheral vasodilator, was FDA approved for the treatment of cerebrovascular insufficiency and peripheral vascular disease. Our demonstration of the significant and novel neuroprotective action of isoxsuprine hydrochloride in an in vivo stroke model and its history of human use suggest that isoxsuprine may be an ideal candidate for further investigation as a potential stroke therapeutic.

Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation.

ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.