Deletion of PTP4A3 phosphatase in high grade serous ovarian cancer cells decreases tumorigenicity and produces marked changes in intracellular signaling pathways and cytokine release.

The oncogenic protein tyrosine phosphatase PTP4A3 is frequently overexpressed in human ovarian cancers and is associated with poor patient prognosis. PTP4A3 is thought to regulate multiple oncogenic signaling pathways, including STAT3, SRC, and ERK. The objective of this study was to generate ovarian cancer cells with genetically depleted PTP4A3; to assess their tumorigenicity; to examine their cellular phenotype; and to uncover changes in their intracellular signaling pathways and cytokine release profiles. Genetic deletion of PTP4A3 using CRISPR/Cas9 enabled the generation of individual clones derived from single cells isolated from the polyclonal knockout population. We observed a >90% depletion of PTP4A3 protein levels by Western blotting in the clonal cell lines compared to the sham transfected wildtype population. The wildtype and polyclonal knockout cell lines shared similar monolayer growth rates, while the isolated clonal populations 2B4, 3C9, and 3C12 exhibited significantly lower monolayer growth characteristics consistent with their lower PTP4A3 levels. The clonal PTP4A3 knockout cell lines also had substantially lower in vitro colony formation efficiencies compared to the wildtype cells and were less tumorigenic in vivo The clonal knockout cells were markedly less responsive to IL-6-stimulated migration in a scratch wound assay compared to the wildtype cells. Antibody microarray assays documented differences in cytokine release and intracellular phosphorylation patterns in the PTP4A3 deleted clones. Bioinformatic network analyses indicated alterations in cellular signaling nodes. These biochemical changes could ultimately form the foundation for pharmacodynamic endpoints useful for emerging anti-PTP4A3 therapeutics. Significance Statement Clones of high grade serous ovarian cancer cells were isolated in which the oncogenic phosphatase PTP4A3 was deleted using CRISPR/Cas9 methodologies. The PTP4A3 null cells exhibited loss of in vitro proliferation, colony formation, and migration, and reduced in vivo tumorigenesis. Marked differences in intracellular protein phosphorylation and cytokine release were seen. The newly developed PTP4A3 knockout cells should provide useful tools to probe the role of PTP4A3 phosphatase in ovarian cancer cell survival, tumorigenicity and cell signaling.

Disruption of Ovarian Cancer STAT3 and p38 Signaling with a Small-Molecule Inhibitor of PTP4A3 Phosphatase.

Protein tyrosine phosphatase type IVA member 3 (PTP4A3 or PRL-3) is a nonreceptor, oncogenic, dual-specificity phosphatase that is highly expressed in many human tumors, including ovarian cancer, and is associated with a poor patient prognosis. Recent studies suggest that PTP4A3 directly dephosphorylates SHP-2 phosphatase as part of a STAT3-PTP4A3 feedforward loop and directly dephosphorylates p38 kinase. The goal of the current study was to examine the effect of a PTP4A phosphatase inhibitor, 7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053), on ovarian cancer STAT3, SHP-2, and p38 kinase phosphorylation. JMS-053 caused a concentration- and time-dependent decrease in the activated form of STAT3, Y705 phospho-STAT3, in ovarian cancer cells treated in vitro. In contrast, the phosphorylation status of two previously described direct PTP4A3 substrates, SHP-2 phosphatase and p38 kinase, were rapidly increased with JMS-053 treatment. We generated A2780 and OVCAR4 ovarian cancer cells resistant to JMS-053, and the resulting cells were not crossresistant to paclitaxel, cisplatin, or teniposide. JMS-053-resistant A2780 and OVCAR4 cells exhibited a 95% and 50% decrease in basal Y705 phospho-STAT3, respectively. JMS-053-resistant OVCAR4 cells had an attenuated phosphorylation and migratory response to acute exposure to JMS-053. These results support a regulatory role for PTP4A phosphatase in ovarian cancer cell STAT3 and p38 signaling circuits. SIGNIFICANCE STATEMENT: This study demonstrates that chemical inhibition of PTP4A phosphatase activity with JMS-053 decreases STAT3 activation and increases SHP-2 phosphatase and p38 kinase phosphorylation activation in ovarian cancer cells. The newly developed JMS-053-resistant ovarian cancer cells should provide useful tools to further probe the role of PTP4A phosphatase in ovarian cancer cell survival and cell signaling.

The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells.

Acute lung injury (ALI) leading to acute respiratory distress syndrome is the major cause of COVID-19 lethality. Cell entry of SARS-CoV-2 occurs via the interaction between its surface spike protein (SP) and angiotensin-converting enzyme-2 (ACE2). It is unknown if the viral spike protein alone is capable of altering lung vascular permeability in the lungs or producing lung injury in vivo. To that end, we intratracheally instilled the S1 subunit of SARS-CoV-2 spike protein (S1SP) in K18-hACE2 transgenic mice that overexpress human ACE2 and examined signs of COVID-19-associated lung injury 72 h later. Controls included K18-hACE2 mice that received saline or the intact SP and wild-type (WT) mice that received S1SP. K18-hACE2 mice instilled with S1SP exhibited a decline in body weight, dramatically increased white blood cells and protein concentrations in bronchoalveolar lavage fluid (BALF), upregulation of multiple inflammatory cytokines in BALF and serum, histological evidence of lung injury, and activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways in the lung. K18-hACE2 mice that received either saline or SP exhibited little or no evidence of lung injury. WT mice that received S1SP exhibited a milder form of COVID-19 symptoms, compared with the K18-hACE2 mice. Furthermore, S1SP, but not SP, decreased cultured human pulmonary microvascular transendothelial resistance (TER) and barrier function. This is the first demonstration of a COVID-19-like response by an essential virus-encoded protein by SARS-CoV-2 in vivo. This model of COVID-19-induced ALI may assist in the investigation of new therapeutic approaches for the management of COVID-19 and other coronaviruses.

Synthesis and evaluation of bifunctional PTP4A3 phosphatase inhibitors activating the ER stress pathway.

We developed JMS-053, a potent inhibitor of the dual specificity phosphatase PTP4A3 that is potentially suitable for cancer therapy. Due to the emerging role of the unfolded protein response (UPR) in cancer pathology, we sought to identify derivatives that combine PTP4A3 inhibition with induction of endoplasmatic reticulum (ER) stress, with the goal to generate more potent anticancer agents. We have now generated bifunctional analogs that link the JMS-053 pharmacophore to an adamantyl moiety and act in concert with the phosphatase inhibitor to induce ER stress and cell death. The most potent compound in this series, 7a, demonstrated a ca. 5-fold increase in cytotoxicity in a breast cancer cell line and strong activation of UPR and ER stress response genes in spite of a ca. 13-fold decrease in PTP4A3 inhibition. These results demonstrate that the combination of phosphatase inhibition with UPR/ER-stress upregulation potentiates efficacy.

Structure of the Complex of an Iminopyridinedione Protein Tyrosine Phosphatase 4A3 Phosphatase Inhibitor with Human Serum Albumin.

Protein tyrosine phosphatase (PTP) 4A3 is frequently overexpressed in human solid tumors and hematologic malignancies and is associated with tumor cell invasion, metastasis, and a poor patient prognosis. Several potent, selective, and allosteric small molecule inhibitors of PTP4A3 were recently identified. A lead compound in the series, JMS-053 (7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione), has a long plasma half-life (∼ 24 hours) in mice, suggesting possible binding to serum components. We confirmed by isothermal titration calorimetry that JMS-053 binds to human serum albumin. A single JMS-053 binding site was identified by X-ray crystallography in human serum albumin at drug site 3, which is also known as subdomain IB. The binding of JMS-053 to human serum albumin, however, did not markedly alter the overall albumin structure. In the presence of serum albumin, the potency of JMS-053 as an in vitro inhibitor of PTP4A3 and human A2780 ovarian cancer cell growth was reduced. The reversible binding of JMS-053 to serum albumin may serve to increase JMS-053's plasma half-life and thus extend the delivery of the compound to tumors. SIGNIFICANCE STATEMENT: X-ray crystallography revealed that a potent, reversible, first-in-class small molecule inhibitor of the oncogenic phosphatase protein tyrosine phosphatase 4A3 binds to at least one site on human serum albumin, which is likely to extend the compound's plasma half-life and thus assist in drug delivery into tumors.

Drugging Undruggable Molecular Cancer Targets.

Cancer, more than any other human disease, now has a surfeit of potential molecular targets poised for therapeutic exploitation. Currently, a number of attractive and validated cancer targets remain outside of the reach of pharmacological regulation. Some have been described as undruggable, at least by traditional strategies. In this article, we outline the basis for the undruggable moniker, propose a reclassification of these targets as undrugged, and highlight three general classes of this imposing group as exemplars with some attendant strategies currently being explored to reclassify them. Expanding the spectrum of disease-relevant targets to pharmacological manipulation is central to reducing cancer morbidity and mortality.

Next-Generation Cell-Active Inhibitors of the Undrugged Oncogenic PTP4A3 Phosphatase.

Oncogenic protein tyrosine phosphatases (PTPs) are overexpressed in numerous human cancers but they have been challenging pharmacological targets. The emblematic oncogenic PTP4A tyrosine phosphatase family regulates many fundamental malignant processes. 7-Imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053) is a novel, potent, and selective PTP4A inhibitor but its mechanism of action has not been fully elucidated, nor has the chemotype been fully investigated. Because tyrosine phosphatases are notoriously susceptible to oxidation, we interrogated JMS-053 and three newly synthesized analogs with specific attention on the role of oxidation. JMS-053 and its three analogs were potent in vitro PTP4A3 inhibitors, but 7-imino-5-methyl-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (NRT-870-59) appeared unique among the thienopyridinediones with respect to its inhibitory specificity for PTP4A3 versus both a PTP4A3 A111S mutant and an oncogenic dual specificity tyrosine phosphatase, CDC25B. Like JMS-053, NRT-870-59 was a reversible PTP4A3 inhibitor. All of the thienopyridinediones retained cytotoxicity against human ovarian and breast cancer cells grown as pathologically relevant three-dimensional spheroids. Inhibition of cancer cell colony formation by NRT-870-59, like JMS-053, required PTP4A3 expression. JMS-053 failed to generate significant detectable reactive oxygen species in vitro or in cancer cells. Mass spectrometry results indicated no disulfide bond formation or oxidation of the catalytic Cys104 after in vitro incubation of PTP4A3 with JMS-053 or NRT-870-59. Gene expression profiling of cancer cells exposed to JMS-053 phenocopied many of the changes seen with the loss of PTP4A3 and did not indicate oxidative stress. These data demonstrate that PTP4A phosphatases can be selectively targeted with small molecules that lack prominent reactive oxygen species generation and encourage further studies of this chemotype. SIGNIFICANCE STATEMENT: Protein tyrosine phosphatases are emerging as important contributors to human cancers. We report on a new class of reversible protein phosphatase small molecule inhibitors that are cytotoxic to human ovarian and breast cancer cells, do not generate significant reactive oxygen species in vitro and in cells, and could be valuable lead molecules for future studies of PTP4A phosphatases.

A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Dysregulation of the tightly controlled protein phosphorylation networks that govern cellular behavior causes cancer. The membrane-associated, intracellular protein tyrosine phosphatase PTP4A3 is overexpressed in human colorectal cancer and contributes to cell migration and invasion. To interrogate further the role of PTP4A3 in colorectal cancer cell migration and invasion, we deleted the Ptp4a3 gene from murine colorectal tumor cells. The resulting PTP4A3-/- cells exhibited impaired colony formation, spheroid formation, migration, and adherence compared with the paired PTP4A3fl/fl cells. We replicated these phenotypic changes using the new small-molecule, allosteric PTP4A3 inhibitor JMS-053. A related structure, JMS-038, which lacked phosphatase inhibition, displayed no cellular activity. Reduction in cell viability and colony formation by JMS-053 occurred in both mouse and human colorectal cell lines and required PTP4A3 expression. Ptp4a3 deletion increased the expression of extracellular matrix (ECM) and adhesion genes, including the tumor suppressor Emilin 1. JMS-053 also increased Emilin 1 gene expression. Moreover, The Cancer Genome Atlas genomic database revealed human colorectal tumors with high Ptp4a3 expression had low Emilin 1 expression. These chemical and biologic reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the ECM and support efforts to pharmacologically target PTP4A3.-McQueeney, K. E., Salamoun, J. M., Ahn J. G., Pekic, P., Blanco, I. K., Struckman, H. L., Sharlow, E. R., Wipf, P., Lazo, J. S. A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Tapping the therapeutic potential of protein tyrosine phosphatase 4A with small molecule inhibitors.

Protein tyrosine phosphatases (PTPs) are emerging new targets for drug discovery. PTPs and protein tyrosine kinases (PTKs) maintain cellular homeostasis through opposing roles: tyrosine O-dephosphorylation and -phosphorylation, respectively. An imbalance in the phosphorylation equilibrium results in aberrant protein signaling and pathophysiological conditions. PTPs have historically been considered 'undruggable', in part due to a lack of evidence defining their relationship to disease causality and a focus on purely competitive inhibitors. However, a better understanding of protein-protein interfaces and shallow active sites has recently renewed interest in the pursuit of allosteric and orthosteric modulators of targets outside the major druggable protein families. While their biological mechanism of action still remains to be clarified, PTP4A1-3 (also referred to as PRL1-3) are validated oncology targets and play an important role in cell proliferation, metastasis, and tumor angiogenesis. In this Digest, recent syntheses and structure-activity relationships (SAR) of small molecule inhibitors (SMIs) of PTP4A1-3 are summarized, and enzyme docking studies of the most potent chemotype are highlighted. In particular, the thienopyridone scaffold has emerged as a potent lead structure to interrogate the function and druggability of this dual-specificity PTP.

In-flow photooxygenation of aminothienopyridinones generates iminopyridinedione PTP4A3 phosphatase inhibitors.

A continuous flow photooxygenation of 7-aminothieno[3,2-c]pyridin-4(5H)-ones to produce 7-iminothieno[3,2-c]pyridine-4,6(5H,7H)-diones has been developed, utilizing ambient air as the sole reactant. N-H Imines are formed as the major products, and excellent functional group tolerance and conversion on gram-scale without the need for chromatographic purification allow for facile late-stage diversification of the aminothienopyridinone scaffold. Several analogs exhibit potent in vitro inhibition of the cancer-associated protein tyrosine phosphatase PTP4A3, and the SAR supports an exploratory docking model.

Antiparasitic lethality of sulfonamidebenzamides in kinetoplastids.

A sulfonamidebenzamide series was assessed for anti-kinetoplastid parasite activity based on structural similarity to the antiparasitic drug, nifurtimox. Through structure-activity optimization, derivatives with limited mammalian cell toxicity and increased potency toward African trypanosomes and Leishmania promastigotes were developed. Compound 22 had the best potency against the trypanosome (EC50=0.010µM) while several compounds showed ∼10-fold less potency against Leishmania promastigotes without impacting mammalian cells (EC50>25µM). While the chemotype originated from an unrelated optimization program aimed at selectively activating an apoptotic pathway in mammalian cancer cells, our preliminary results suggest that a distinct mechanism of action from that observed in mammalian cells is responsible for the promising activity observed in parasites.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 µM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.

Photooxygenation of an amino-thienopyridone yields a more potent PTP4A3 inhibitor.

The phosphatase PTP4A3 is an attractive anticancer target, but knowledge of its exact role in cells remains incomplete. A potent, structurally novel inhibitor of the PTP4A family was obtained by photooxygenation of a less active, electron-rich thienopyridone (1). Iminothienopyridinedione 13 displays increased solution stability and is readily obtained by two new synthetic routes that converge in the preparation of 1. The late-stage photooxygenation of 1 to give 13 in high yield highlights the potential of this reaction to modify the structure and properties of a biological lead compound and generate value for expanding the scope of an SAR investigation. Analog 13 should become a valuable tool for further exploration of the role of PTP4A3 in tumor progression.

Chemical genetics of Plasmodium falciparum.

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library-many of which showed potent in vitro activity against drug-resistant P. falciparum strains-and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.

Synthesis and biological evaluation of 3-aminoisoquinolin-1(2H)-one based inhibitors of the dual-specificity phosphatase Cdc25B.

The cell division cycle 25B dual specificity phosphatase (Cdc25B) regulates the normal progression of the mammalian cell cycle by dephosphorylating and activating cyclin-dependent kinase (Cdk) complexes, particularly in response to DNA damage. Elevated Cdc25B levels enable a bypass of normal cell cycle checkpoints, and the overexpression of Cdc25B has been linked to a variety of human cancers. Thus, Cdc25B is an attractive target for the development of anticancer therapeutics. Herein we describe the synthesis and biological evaluation of a series of non-quinoid inhibitors of Cdc25B containing the 3-aminoisoquinolin-1(2H)-one pharmacophore. In addition to several strategies that address specific substitution patterns on isoquinolines, we have applied a regioselective Pd-catalyzed cross-coupling methodology to synthesize a new lead structure, 6-(3-aminophenyl)-3-(phenylamino)isoquinolin-1(2H)-one (13), which proved to be a reversible, competitive Cdc25B inhibitor with a Ki of 1.9µM. Compound 13 prevented human cancer cell growth and blocked Cdc25B-mediated mitotic checkpoint bypass. Molecular docking studies support binding near the catalytic site.

Pharmacologic profiling of phosphoinositide 3-kinase inhibitors as mitigators of ionizing radiation-induced cell death.

Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death.

Pharmacological profiling identifies divergent chemosensitivities of differentiating and maturing iPSC-derived human cortical neuron populations.

Neuronal differentiation and maturation are extended developmental processes. To determine whether neurons at different developmental stages have divergent chemosensitivities, we screened differentiating and maturing neuronal populations using a small compound library comprising FDA-approved and investigational drugs. Using a neurotoxicity assay format, both respective neuronal population-based screening campaigns performed robustly (Z-factors = 0.7-0.8), although the hit rate for the differentiating neurons (2.8%) was slightly higher than for maturing neurons (1.9%). While the majority of hits were toxic to both neuronal populations, these hits predominantly represented promiscuous drugs. Other drugs were selectively neurotoxic, with receptor tyrosine kinase inhibitors disproportionally represented after confirmation. Ponatinib and amuvatinib were neuroinhibitory for differentiating and maturing neurons, respectively. Chemoinformatic analyses confirmed differences in potential drug targets that may be differentially expressed during neuronal development. Subsequent studies demonstrated neuronal expression of AXL, an amuvatinib target, in both neuronal populations. However, functional AXL activity was confirmed only in the maturing neuronal population as determined by AXL phosphorylation in response to GAS6, the cognate ligand of AXL, and concurrent STAT3Y705 phosphorylation. Differentiating neurons were unresponsive to the effects of GAS6 suggesting that the AXL-STAT3 signaling axis was nonfunctional. Amuvatinib treatment of maturing neuronal cultures significantly reduced pAXL levels. These studies indicate that neuronal developmental states may exhibit unique chemosensitivities and that drugs may have different neuro-inhibitory effects depending upon the developmental stage of the neuronal population.

High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, iPSC-derived neuronal differentiation and maturation is time-intensive, often requiring >8 weeks. Unfortunately, the differentiating and maturing iPSC-derived neuronal cultures also tend to migrate and coalesce into ganglion-like clusters making single-cell analysis challenging, especially in miniaturized formats. Using our defined extracellular matrix and low oxygen culturing conditions for the differentiation and maturation of human cortical neurons, we further modified neuronal progenitor cell seeding densities and feeder layer-free culturing conditions in miniaturized formats (i.e., 96 well) to decrease neuronal clustering, enhance single-cell identification and reduce edge effects usually observed after extended neuronal cell culture. Subsequent algorithm development refined capabilities to distinguish and identify single mature neurons, as identified by NeuN expression, from large cellular aggregates, which were excluded from image analysis. Incorporation of astrocyte conditioned medium during differentiation and maturation periods significantly increased the percentage (i.e., ∼10% to ∼30%) of mature neurons (i.e., NeuN+) detected at 4-weeks post-differentiation. Pilot, proof of concept studies using this optimized assay system yielded negligible edge effects and robust Z-factors in population-based as well as image-based neurotoxicity assay formats. Moreover, moxidectin, an FDA-approved drug with documented neurotoxic adverse effects, was identified as a hit using both screening formats. This miniaturized, feeder layer-free format and image analysis algorithm provides a foundational imaging and screening platform, which enables quantitative single-cell analysis of differentiated human neurons.

Transcriptome and Translatome Regulation of Pathogenesis in Alzheimer's Disease Model Mice.

BACKGROUND: Defining cellular mechanisms that drive Alzheimer's disease (AD) pathogenesis and progression will be aided by studies defining how gene expression patterns change during pre-symptomatic AD and ensuing periods of declining cognition. Previous studies have emphasized changes in transcriptome, but not translatome regulation, leaving the ultimate results of gene expression alterations relatively unexplored in the context of AD. OBJECTIVE: To identify genes whose expression might be regulated at the transcriptome and translatome levels in AD, we analyzed gene expression in cerebral cortex of two AD model mouse strains, CVN (APPSwDI;NOS2 -/- ) and Tg2576 (APPSw), and their companion wild type (WT) strains at 6 months of age by tandem RNA-Seq and Ribo-Seq (ribosome profiling). METHODS: Identical starting pools of bulk RNA were used for RNA-Seq and Ribo-Seq. Differential gene expression analysis was performed at the transcriptome, translatome, and translational efficiency levels. Regulated genes were functionally evaluated by gene ontology tools. RESULTS: Compared to WT mice, AD model mice had similar levels of transcriptome regulation, but differences in translatome regulation. A microglial signature associated with early stages of Aß accumulation was upregulated at both levels in CVN mice. Although the two mice strains did not share many regulated genes, they showed common regulated pathways related to AßPP metabolism associated with neurotoxicity and neuroprotection. CONCLUSION: This work represents the first genome-wide study of brain translatome regulation in animal models of AD and provides evidence of a tight and early translatome regulation of gene expression controlling the balance between neuroprotective and neurodegenerative processes in brain.

Protein kinase D as a potential new target for cancer therapy.

Protein kinase D is a novel family of serine/threonine kinases and diacylglycerol receptors that belongs to the calcium/calmodulin-dependent kinase superfamily. Evidence has established that specific PKD isoforms are dysregulated in several cancer types, and PKD involvement has been documented in a variety of cellular processes important to cancer development, including cell growth, apoptosis, motility, and angiogenesis. In light of this, there has been a recent surge in the development of novel chemical inhibitors of PKD. This review focuses on the potential of PKD as a chemotherapeutic target in cancer treatment and highlights important recent advances in the development of PKD inhibitors.