Concentration and proteolysis of CX3CL1 may regulate the microglial response to CX3CL1.

Fractalkine (FKN) is a membrane-bound chemokine that can be cleaved by proteases such as ADAM 10, ADAM 17, and cathepsin S to generate soluble fragments. Studies using different forms of the soluble FKN yield conflicting results in vivo. These observations prompted us to investigate the function and pharmacology of two commonly used isoforms of FKN, a human full-length soluble FKN (sFKN), and a human chemokine domain only FKN (cdFKN). Both are prevalent in the literature and are often assumed to be functionally equivalent. We observed that recombinant sFKN and cdFKN exhibit similar potencies in a cell-based cAMP assay, but binding affinity for CX3CR1 was modestly different. There was a 10-fold difference in potency between sFKN and cdFKN when assessing their ability to stimulate ß-arrestin recruitment. Interestingly, high concentrations of FKN, regardless of cleavage variant, were ineffective at reducing pro-inflammatory microglial activation and may induce a pro-inflammatory response. This effect was observed in mouse and rat primary microglial cells as well as microglial cell lines. The inflammatory response was exacerbated in aged microglia, which is known to exhibit age-related inflammatory phenotypes. We observed the same effects in Cx3cr1-/- primary microglia and therefore speculate that an alternative FKN receptor may exist. Collectively, these data provide greater insights into the function and pharmacology of these common FKN reagents, which may clarify conflicting reports and urge greater caution in the selection of FKN peptides for use in in vitro and in vivo studies and the interpretation of results obtained using these differing peptides.

Optimization of a urea-containing series of nicotinamide phosphoribosyltransferase (NAMPT) activators.

NAD+ is a crucial cellular factor that plays multifaceted roles in wide ranging biological processes. Low levels of NAD+ have been linked to numerous diseases including metabolic disorders, cardiovascular disease, neurodegeneration, and muscle wasting disorders. A novel strategy to boost NAD+ is to activate nicotinamide phosphoribosyltransferase (NAMPT), the putative rate-limiting step in the NAD+ salvage pathway. We previously showed that NAMPT activators increase NAD+ levels in vitro and in vivo. Herein we describe the optimization of our NAMPT activator prototype (SBI-0797812) leading to the identification of 1-(4-((4-chlorophenyl)sulfonyl)phenyl)-3-(oxazol-5-ylmethyl)urea (34) that showed far more potent NAMPT activation and improved oral bioavailability.

Discovery of small molecule antagonists of chemokine receptor CXCR6 that arrest tumor growth in SK-HEP-1 mouse xenografts as a model of hepatocellular carcinoma.

The chemokine system plays an important role in mediating a proinflammatory microenvironment for tumor growth in hepatocellular carcinoma (HCC). The CXCR6 receptor and its natural ligand CXCL16 are expressed at high levels in HCC cell lines and tumor tissues and receptor expression correlates with increased neutrophils in these tissues contributing to poor prognosis in patients. Availability of pharmacologcal tools targeting the CXCR6/CXCL16 axis are needed to elucidate the mechanism whereby neutrophils are affected in the tumor environment. We report the discovery of a series of small molecules with an exo-[3.3.1]azabicyclononane core. Our lead compound 81 is a potent (EC50 = 40 nM) and selective orally bioavailable small molecule antagonist of human CXCR6 receptor signaling that significantly decreases tumor growth in a 30-day mouse xenograft model of HCC.

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11.

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

Discovery of Novel Small-Molecule Inducers of Heme Oxygenase-1 That Protect Human iPSC-Derived Cardiomyocytes from Oxidative Stress.

Oxidative injury to cardiomyocytes plays a critical role in cardiac pathogenesis following myocardial infarction. Transplantation of stem cell-derived cardiomyocytes has recently progressed as a novel treatment to repair damaged cardiac tissue but its efficacy has been limited by poor survival of transplanted cells owing to oxidative stress in the post-transplantation environment. Identification of small molecules that activate cardioprotective pathways to prevent oxidative damage and increase survival of stem cells post-transplantation is therefore of great interest for improving the efficacy of stem cell therapies. This report describes a chemical biology phenotypic screening approach to identify and validate small molecules that protect human-induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from oxidative stress. A luminescence-based high-throughput assay for cell viability was used to screen a diverse collection of 48,640 small molecules for protection of hiPSC-CMs from peroxide-induced cell death. Cardioprotective activity of "hit" compounds was confirmed using impedance-based detection of cardiomyocyte monolayer integrity and contractile function. Structure-activity relationship studies led to the identification of a potent class of compounds with 4-(pyridine-2-yl)thiazole scaffold. Examination of gene expression in hiPSC-CMs revealed that the hit compound, designated cardioprotectant 312 (CP-312), induces robust upregulation of heme oxygenase-1, a marker of the antioxidant response network that has been strongly correlated with protection of cardiomyocytes from oxidative stress. CP-312 therefore represents a novel chemical scaffold identified by phenotypic high-throughput screening using hiPSC-CMs that activates the antioxidant defense response and may lead to improved pharmacological cardioprotective therapies.

Oleoylethanolamide modulates glucagon-like peptide-1 receptor agonist signaling and enhances exendin-4-mediated weight loss in obese mice.

Long-acting glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists (GLP-1RA), such as exendin-4 (Ex4), promote weight loss. On the basis of a newly discovered interaction between GLP-1 and oleoylethanolamide (OEA), we tested whether OEA enhances GLP-1RA-mediated anorectic signaling and weight loss. We analyzed the effect of GLP-1+OEA and Ex4+OEA on canonical GLP-1R signaling and other proteins/pathways that contribute to the hypophagic action of GLP-1RA (AMPK, Akt, mTOR, and glycolysis). We demonstrate that OEA enhances canonical GLP-1R signaling when combined with GLP-1 but not with Ex4. GLP-1 and Ex4 promote phosphorylation of mTOR pathway components, but OEA does not enhance this effect. OEA synergistically enhanced GLP-1- and Ex4-stimulated glycolysis but did not augment the hypophagic action of GLP-1 or Ex4 in lean or diet-induced obese (DIO) mice. However, the combination of Ex4+OEA promoted greater weight loss in DIO mice than Ex4 or OEA alone during a 7-day treatment. This was due in part to transient hypophagia and increased energy expenditure, phenotypes also observed in Ex4-treated DIO mice. Thus, OEA augments specific GLP-1RA-stimulated signaling but appears to work in parallel with Ex4 to promote weight loss in DIO mice. Elucidating cooperative mechanisms underlying Ex4+OEA-mediated weight loss could, therefore, be leveraged toward more effective obesity therapies.

APJ acts as a dual receptor in cardiac hypertrophy.

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of ß-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.

Assessment of drug-induced arrhythmic risk using limit cycle and autocorrelation analysis of human iPSC-cardiomyocyte contractility.

Cardiac safety assays incorporating label-free detection of human stem-cell derived cardiomyocyte contractility provide human relevance and medium throughput screening to assess compound-induced cardiotoxicity. In an effort to provide quantitative analysis of the large kinetic datasets resulting from these real-time studies, we applied bioinformatic approaches based on nonlinear dynamical system analysis, including limit cycle analysis and autocorrelation function, to systematically assess beat irregularity. The algorithms were integrated into a software program to seamlessly generate results for 96-well impedance-based data. Our approach was validated by analyzing dose- and time-dependent changes in beat patterns induced by known proarrhythmic compounds and screening a cardiotoxicity library to rank order compounds based on their proarrhythmic potential. We demonstrate a strong correlation for dose-dependent beat irregularity monitored by electrical impedance and quantified by autocorrelation analysis to traditional manual patch clamp potency values for hERG blockers. In addition, our platform identifies non-hERG blockers known to cause clinical arrhythmia. Our method provides a novel suite of medium-throughput quantitative tools for assessing compound effects on cardiac contractility and predicting compounds with potential proarrhythmia and may be applied to in vitro paradigms for pre-clinical cardiac safety evaluation.

Exendin-4 improves cardiac function in mice overexpressing monocyte chemoattractant protein-1 in cardiomyocytes.

The incretin hormone glucagon-like peptide-1 (Glp1) is cardioprotective in models of ischemia-reperfusion injury, myocardial infarction and gluco/lipotoxicity. Inflammation is a factor in these models, yet it is unknown whether Glp1 receptor (Glp1r) agonists are protective against cardiac inflammation. We tested the hypothesis that the Glp1r agonist Exendin-4 (Ex4) is cardioprotective in mice with cardiac-specific monocyte chemoattractant protein-1 overexpression. These MHC-MCP1 mice exhibit increased cardiac monocyte infiltration, endoplasmic reticulum (ER) stress, apoptosis, fibrosis and left ventricular dysfunction. Ex4 treatment for 8 weeks improved cardiac function and reduced monocyte infiltration, fibrosis and apoptosis in MHC-MCP1 mice. Ex4 enhanced expression of the ER chaperone glucose-regulated protein-78 (GRP78), decreased expression of the pro-apoptotic ER stress marker CCAAT/-enhancer-binding protein homologous protein (CHOP) and increased expression of the ER calcium regulator Sarco/Endoplasmic Reticulum Calcium ATPase-2a (SERCA2a). These findings suggest that the Glp1r is a viable target for treating cardiomyopathies associated with stimulation of pro-inflammatory factors.

Plasminogen activator inhibitor-1 antagonist TM5441 attenuates Nω-nitro-L-arginine methyl ester-induced hypertension and vascular senescence.

BACKGROUND: Long-term inhibition of nitric oxide synthase by L-arginine analogues such as N(ω)-nitro-l-arginine methyl ester (L-NAME) has been shown to induce senescence in vitro and systemic hypertension and arteriosclerosis in vivo. We previously reported that plasminogen activator inhibitor-1 (PAI-1)-deficient mice (PAI-1(-/-)) are protected against L-NAME-induced pathologies. In this study, we investigated whether a novel, orally active PAI-1 antagonist (TM5441) has a similar protective effect against L-NAME treatment. Additionally, we studied whether L-NAME can induce vascular senescence in vivo and investigated the role of PAI-1 in this process. METHODS AND RESULTS: Wild-type mice received either L-NAME or L-NAME and TM5441 for 8 weeks. Systolic blood pressure was measured every 2 weeks. We found that TM5441 attenuated the development of hypertension and cardiac hypertrophy compared with animals that had received L-NAME alone. Additionally, TM5441-treated mice had a 34% reduction in periaortic fibrosis relative to animals on L-NAME alone. Finally, we investigated the development of vascular senescence by measuring p16(Ink4a) expression and telomere length in aortic tissue. We found that L-NAME increased p16(Ink4a) expression levels and decreased telomere length, both of which were prevented with TM5441 cotreatment. CONCLUSIONS: Pharmacological inhibition of PAI-1 is protective against the development of hypertension, cardiac hypertrophy, and periaortic fibrosis in mice treated with L-NAME. Furthermore, PAI-1 inhibition attenuates the arterial expression of p16(Ink4a) and maintains telomere length. PAI-1 appears to play a pivotal role in vascular senescence, and these findings suggest that PAI-1 antagonists may provide a novel approach in preventing vascular aging and hypertension.

Targeting the IRE1α/XBP1 and ATF6 arms of the unfolded protein response enhances VEGF blockade to prevent retinal and choroidal neovascularization.

Although anti-vascular endothelial growth factor (VEGF) treatments reduce pathological neovascularization in the eye and in tumors, the regression is often not sustainable or is incomplete. We investigated whether vascular endothelial cells circumvent anti-VEGF therapies by activating the unfolded protein response (UPR) to override the classic extracellular VEGF pathway. Exposure of endothelial cells to VEGF, high glucose, or H2O2 up-regulated the X-box binding protein-1/inositol-requiring protein-1 (IRE1) α and activating transcription factor 6 (ATF6) arms of the UPR compared with untreated cells. This was associated with increased expression in α-basic crystallin (CRYAB), which has previously bound VEGF. siRNA knockdown or pharmacological blockade of IRE1α, ATF6, or CRYAB increased intracellular VEGF degradation and decreased full-length intracellular VEGF. Inhibition of IRE1α, ATF6, or CRYAB resulted in an approximately 40% reduction of in vitro angiogenesis, which was further reduced in combination with a neutralizing antibody against extracellular VEGF. Blockade of IRE1α or ATF6 in the oxygen-induced retinopathy or choroidal neovascularization mouse models caused an approximately 35% reduction in angiogenesis. However, combination therapy of VEGF neutralizing antibody with UPR inhibitors or siRNAs reduced retinal/choroidal neovascularization by a further 25% to 40%, and this inhibition was significantly greater than either treatment alone. In conclusion, activation of the UPR sustains angiogenesis by preventing degradation of intracellular VEGF. The IRE1α/ATF6 arms of the UPR offer a potential therapeutic target in the treatment of pathological angiogenesis.

Design, synthesis and evaluation of benzoisothiazolones as selective inhibitors of PHOSPHO1.

We report the discovery and characterization of a series of benzoisothiazolone inhibitors of PHOSPHO1, a newly identified soluble phosphatase implicated in skeletal mineralization and soft tissue ossification abnormalities. High-throughput screening (HTS) of a small molecule library led to the identification of benzoisothiazolones as potent and selective inhibitors of PHOSPHO1. Critical structural requirements for activity were determined, and the compounds were subsequently derivatized and measured for in vitro activity and ADME parameters including metabolic stability and permeability. On the basis of its overall profile the benzoisothiazolone analogue 2q was selected as MLPCN probe ML086.

Imidazole-derived agonists for the neurotensin 1 receptor.

A scaffold-hop program seeking full agonists of the neurotensin-1 (NTR1) receptor identified the probe molecule ML301 (1) and associated analogs, including its naphthyl analog (14) which exhibited similar properties. Compound 1 showed full agonist behavior (79-93%) with an EC50 of 2.0-4.1µM against NTR1. Compound 1 also showed good activity in a Ca mobilization FLIPR assay (93% efficacy at 298nM), consistent with it functioning via the Gq coupled pathway, and good selectivity relative to NTR2 and GPR35. In further profiling, 1 showed low potential for promiscuity and good overall pharmacological data. This report describes the discovery, synthesis, and SAR of 1 and associated analogs. Initial in vitro pharmacologic characterization is also presented.

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor.

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ~330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 µM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.

Antidiabetic drug metformin (GlucophageR) increases biogenesis of Alzheimer's amyloid peptides via up-regulating BACE1 transcription.

Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of beta-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of beta-amyloid (Abeta) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage(R)), affects APP metabolism and Abeta generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular Abeta species. Furthermore, the effect of metformin on Abeta generation is mediated by transcriptional up-regulation of beta-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on Abeta degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on Abeta. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on Abeta generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on Abeta generation, in combined use, metformin enhances insulin's effect in reducing Abeta levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients.

Novel and Structurally Diversified Bacterial DNA Gyrase Inhibitors Discovered through a Fluorescence-Based High-Throughput Screening Assay.

Bacterial DNA gyrase, a type IIA DNA topoisomerase that plays an essential role in bacterial DNA replication and transcription, is a clinically validated target for discovering and developing new antibiotics. In this article, based on a supercoiling-dependent fluorescence quenching (SDFQ) method, we developed a high-throughput screening (HTS) assay to identify inhibitors targeting bacterial DNA gyrase and screened the National Institutes of Health's Molecular Libraries Small Molecule Repository library containing 370,620 compounds in which 2891 potential gyrase inhibitors have been identified. According to these screening results, we acquired 235 compounds to analyze their inhibition activities against bacterial DNA gyrase using gel- and SDFQ-based DNA gyrase inhibition assays and discovered 155 new bacterial DNA gyrase inhibitors with a wide structural diversity. Several of them have potent antibacterial activities. These newly discovered gyrase inhibitors include several DNA gyrase poisons that stabilize the gyrase-DNA cleavage complexes and provide new chemical scaffolds for the design and synthesis of bacterial DNA gyrase inhibitors that may be used to combat multidrug-resistant bacterial pathogens. Additionally, this HTS assay can be applied to screen inhibitors against other DNA topoisomerases.

Development and use of a high-throughput screen to identify novel modulators of the corticotropin releasing factor binding protein.

BACKGROUND: Stress responses are believed to involve corticotropin releasing factor (CRF), its two cognate receptors (CRF1 and CRF2), and the CRF-binding protein (CRFBP). Whereas decades of research has focused on CRF1, the role of CRF2 in the central nervous system (CNS) has not been thoroughly investigated. We have previously reported that CRF2, interacting with a C terminal fragment of CRFBP, CRFBP(10kD), may have a role in the modulation of neuronal activity. However, the mechanism by which CRF interacts with CRFBP(10kD) and CRF2 has not been fully elucidated due to the lack of useful chemical tools to probe CRFBP. METHODS: We miniaturized a cell-based assay, where CRFBP(10kD) is fused as a chimera with CRF2, and performed a high-throughput screen (HTS) of 350,000 small molecules to find negative allosteric modulators (NAMs) of the CRFBP(10kD)-CRF2 complex. Hits were confirmed by evaluating activity toward parental HEK293 cells, toward CRF2 in the absence of CRFBP(10kD), and toward CRF1 in vitro. Hits were further characterized in ex vivo electrophysiology assays that target: 1) the CRF1+ neurons in the central nucleus of the amygdala (CeA) of CRF1:GFP mice that express GFP under the CRF1 promoter, and 2) the CRF-induced potentiation of N-methyl-D-aspartic acid receptor (NMDAR)-mediated synaptic transmission in dopamine neurons in the ventral tegmental area (VTA). RESULTS: We found that CRFBP(10kD) potentiates CRF-intracellular Ca2+ release specifically via CRF2, indicating that CRFBP may possess excitatory roles in addition to the inhibitory role established by the N-terminal fragment of CRFBP, CRFBP(27kD). We identified novel small molecule CRFBP-CRF2 NAMs that do not alter the CRF1-mediated effects of exogenous CRF but blunt CRF-induced potentiation of NMDAR-mediated synaptic transmission in dopamine neurons in the VTA, an effect mediated by CRF2 and CRFBP. CONCLUSION: These results provide the first evidence of specific roles for CRF2 and CRFBP(10kD) in the modulation of neuronal activity and suggest that CRFBP(10kD)-CRF2 NAMs can be further developed for the treatment of stress-related disorders including alcohol and substance use disorders.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

Image analysis algorithms for immunohistochemical assessment of cell death events and fibrosis in tissue sections.

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections.

Discovery of ß-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators.

Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.