1,721,037 research outputs found
Academic drug discovery in an age of research abundance, and the curious case of chemical screens toward drug repositioning
No full textHigh-throughput screening (HTS) is a vaunted technology in drug discovery, and drug repositioning a celebrated strategy with famous examples of successful stories; however, repositioned drugs have primarily resulted from serendipitous observations, retrospective studies, and pharmacological analyses as opposed to experimental routes. This observation points to a methodological paradox, considering that academic laboratories of the postgenomic era have benefited from unprecedented technological progress, and a facilitated access to powerful resources that, historically, were a prerogative of the pharma industry. This disconnect is exacerbated by financial, practical, and regulatory complexities affecting drug repositioning; however, the pivotal significance of stringent and rigorous data is what unconditionally sits at the crossroad of go/no-go decisions concerning the therapeutic significance, or predictive validity, of selected drugs. Here, I propose a visionary approach, to which I assigned the term labsourcing, to dramatically enhance efficiency and clinical relevance of academic drug screens and, ultimately, generate contextual and reproducible data for correct interpretations and reliable selection of drug candidates. The overall concept implies intra- and intermural aggregation of expertise (e.g., assay development, cell biology, statistics, bioinformatics) to perform multiple bioassays, under multiple conditions and readouts, using a common screening collection. Advantages of high input screens can be manifold: (i) to tackle discrepancies that may arise from the screens of libraries of variable size and content and assay types and conditions too narrow in scope; (ii) the opportunity to generate massive amounts of data applicable for multiple publications and funding requests; (iii) the educational benefits for students and post-docs collegially exposed to long-term programs; and (iv) the opportunity to democratize research and recruit small labs that could not otherwise join screening programs due to costs, timelines, and risks
Regulation of UDP-GlcNAc:Galβ1-3GalNAc-R β 1-6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) in Chinese hamster ovary cells
No full textUDP-GlcNAc:Galbeta3GalNAc-R (GlcNAc to GalNAc) beta1-6-N-acetylglucosaminyltransferase (i.e. core 2 GlcNAc-T) of the O-linked oligosaccharide pathway is developmentally regulated in human T cells, and changes in its activity have been associated with malignancies and the Wiskott-Aldrich immunodeficiency syndrome. Chinese hamster ovary (CHO) cells normally express low levels of core 2 GlcNAc-T activity (8-12 pmol/mg/h) which can be accurately measured with a two-step assay employing purified bovine
beta1-4Gal-T and high specific activity UDP-[3H]Gal to radiolabel the core 2 reaction product. CHO cells treated with 2 mM sodium butyrate for 24 h exhibited a 16-fold increase in core 2 GlcNAc-T activity, whereas several other differentiating agents including dimethyl
sulfoxide, retinoic acid, phorbol ester, and cholera toxin had no effect on activity. The addition of butyrate, cholera toxin, or dimethyl sulfoxide to CHO cells slowed cell proliferation and induced changes in cell morphology characteristic of cell differentiation. Induction of core 2 GlcNAc-T by butyrate was blocked by actinomycin D and cycloheximide. Butyrate treatment also elevated cytosolic cAMP levels with a time course which paralleled, but preceded, induction of core 2 GlcNAc-T activity by approximately 8 h. The protein kinase inhibitors H-7 and H-8 blocked butyrate-dependent induction of enzyme activity, whereas the inactive analogue H1004 had no effect. Core 2
GlcNAc-T showed a change in Km for UDP-GlcNAc, from 0.50 mM in untreated cells to 4.54 mM in butyrate + cholera toxin treated CHO cells, but no changes in Km for the synthetic acceptor, Galbeta1-3GalNAcalpha-paranitrophenyl. Despite the 9-fold increase in Km for
sugar nucleotide, Vmax/Km was 8.8-fold greater in treated compared with untreated cells. These observations suggest that in CHO cells induction of core 2 GlcNAc-T by butyrate treatment requires de novo gene transcription/translation, activation of protein kinase(s), and is associated with changes in the kinetic properties of the enzyme
β1,6 N-Acetylglucosaminyltransferase (core 2 GlcNAc-T) expression in normal rat tissues and different cell lines: evidence for complex mechanisms of regulation
No full textThe distribution of the Golgi enzyme beta1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T for short) has been investigated in several tissue and cell systems by combining the
potentials of a polyclonal antibody and a novel, sensitive fluorescent
enzyme assay. In normal rat tissues, levels of the protein were found to vary and as a general trend did not correlate with enzyme activities. Additionally, we observed tissue-specific core 2 GlcNAc-T forms of various size: 75 kDa (liver), 70 kDa (spleen), 60 kDA (heart), and 50 kDa (heart and lung). These forms might arise from differential protein modifications; alternatively, the smaller form may be a product of proteolytic cleavage, given the presence of a catalytically inactive 50 kDa species in rat serum. Chinese hamster
ovary (CHO), MDAY-D2, PSA-5E, and PYS-2 cell lines consistently
displayed a 70 kDa enzyme. When induced to retrodifferentiate in the presence of butyrate + cholera toxin, CHO cells exhibited a 21-fold increase in enzyme activity, while protein levels remained constant. A similar trend was observed in the embryonal endoderm cell lines PSA-5E and PYS-2, where an approximately 100-fold difference in core 2
GlcNAc-T activity was found notwithstanding unchanged amounts of the protein and identical mRNA levels, as evidenced by RT-PCR. In contrast, levels of core 2 GlcNAc-T activity in MDAY-D2 cells correlated well with protein expression. Taken together, these observations demonstrate that core 2 GlcNAc-T expression may be subjected to multiple mechanisms of regulation and suggest that in at least some instances (i.e., PSA-5E and PYS-2 cells) expression may be
regulated exclusively via posttranslational mechanism(s) of
control
High throughput kinase inhibitor screens reveal TRB3 and MAPK-ERK/TGF beta pathways as fundamental Notch regulators in breast cancer
No full textExpression of the Notch ligand Jagged 1 (JAG1) and Notch activation promote poor prognosis in breast cancer. We used high throughput screens to identify elements responsible for Notch activation in this context. Chemical kinase inhibitor and kinase-specific small interfering RNA libraries were screened in a breast cancer cell line engineered to report Notch. Pathway analyses revealed MAPK-ERK signaling to be the predominant JAG1/Notch regulator and this was supported by gene set enrichment analyses in 51 breast cancer cell lines. In accordance with the chemical screen, kinome small interfering RNA high throughput screens identified Tribbles homolog 3
(TRB3), a known regulator of MAPK-ERK, among the most significant hits. We demonstrate that TRB3 is a master regulator of Notch through the MAPK-ERK and TGFβ pathways. Complementary in vitro and in vivo studies underscore the importance of TRB3 for tumor growth. These data demonstrate a dominant role for TRB3 and MAPK-ERK/TGFβ pathways as Notch regulators in breast cancer,
establishing TRB3 as a potential therapeutic target
Calcium ionophore A-23187 inhibits the secretion of β-hexosaminidase from the GG2EE mouse macrophage cell line
No full textSecretion of the lysosomal enzyme β-N-acetylhexosaminidase is inhibited by calcium ionophore A-23187 in the GG2EE macrophage cell line. Such inhibition is time and dose dependent. Calcium ionophore A-23187 treatment causes a change in the pattern of hexosaminidase isoenzymes detectable in the cell extract, as assessed by DEAE-cellulose chromatography. In particular, control cells show two hexosaminidase isoenzymes corresponding to hexosaminidase A and B, whereas cells treated with calcium ionophore A-23187 express a third isoenzyme form with properties similar to hexosaminidase S
A HOMOGENEOUS CELL-BASED ASSAY TO IDENTIFY N-LINKED CARBOHYDRATE PROCESSING INHIBITORS
No full textMalignant transformation is accompanied by altered cell surface glycosylation. N-Linked oligosaccharides carrying β1–6GlcNAc branches are associated with tumor invasion and metastasis. Therefore, compounds that can enter cells and block biosynthesis of β1–6GlcNAc-branched glycans without overt cytotoxicity are potential anticancer agents. We have developed a homogeneous cell-based assay for detection of such compounds. The method enables identification of agents that block β1–6GlcNAc-branched glycan expression after incubation for 16–20 h with MDAY-D2 tumor cells, thereby protecting the cells from the subsequent addition of leukoagglutinin, a cytotoxic plant lectin. We observed that MDAY-D2 cell number is directly proportional to the level of endogenous alkaline phosphatase activity measured spectrophotometrically in cultures after the addition of substrate. The alkaline phosphatase assay was capable of detecting as few as 1500 cells. The assay was readily adapted for high-throughput screening as reagent costs are low and no cell harvesting and washing steps are required. Under high-throughput operating conditions, the coefficient of variation for controls was found to be 4.2%. The results suggest that measurement of alkaline phosphatase in this cell assay format may be adapted for wider applications in high-throughput screenings for compounds that relieve cells from other growth inhibitors
COX inhibitors and bone: A safer impact on osteoblasts by NO-releasing NSAIDs
No full textNonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for the treatment of pain and inflammation. Although it is well known that NSAIDs can suppress bone growth, remodelling and repair, they are largely used post-operatively and post-traumatically to achieve analgesia and reduce inflammation in bone tissue. Aims: The impact of two NO-releasing, non-selective NSAIDs, NCX-4016 and HCT-3012 (NO-derivatives of Aspirin and Naproxen, respectively) on osteoblasts were evaluated and compared to the non-selective, parent chemicals and to the COX-2-selective inhibitor Celecoxib. Main methods: Using MG-63 osteoblast-like cells, we considered proliferation, the early and late stage of differentiation, and the activity of proteinases thought to be involved in osteoid degradation, a preliminary fundamental event of bone remodelling. Key findings: Unlike Aspirin, Naproxen and Celecoxib, the two NO-NSAIDs did not alter proliferation and differentiation of osteoblasts. They also reduced the activity of plasminogen activator, metalloproteinases, and cathepsin B. Similar inhibitory effects against these proteinases were recapitulated by the NO-donor sodium nitroprusside, thereby suggesting a NO-mediated mechanism. Significance: Due to a differential effect on cell proliferation and differentiation, the two NO-NSAIDs exhibit a safer impact on osteoblast metabolism compared to Celecoxib and their parent compounds. This suggests an advantageous option for these drugs in individuals with a need of COX-inhibiting treatment, in general. In addition, their capability of modulating the proteinases involved in osteoid degradation may specifically suggest an additional safer use in comorbidity conditions of inflammation or pain with bone disorders characterized by high rate of remodelling, such as high-turnover osteoporosis in post-menopausal women
Luteolin inhibition of V-ATPase a3-d2 interaction decreases osteoclast resorptive activity
No full textV-ATPase-mediated acid secretion is required for osteoclast bone resorption. Osteoclasts are enriched in V-ATPase a3 and d2 subunit isoforms, and disruption of either of their genes impairs bone resorption. Using purified fusion proteins of a3 N-terminal domain (NTa3) and full-length d subunits we determined in a solid-phase binding assay that half-maximal binding of d1 or d2 to immobilized NTa3 occurs at 3.1 ± 0.4 or 3.6 ± 0.6 nM, respectively, suggesting equally high-affinity interactions. A high-throughput modification of this assay was then used to screen chemical libraries for a3–d2 interaction inhibitors, and luteolin, a naturally occurring flavonoid, was identified, with half-maximal inhibition at 2.4 ± 0.9 μM. Luteolin did not significantly affect NIH/3T3 or RAW 264.7 cell viability, nor did it affect cytokine-induced osteoclastogenesis of RAW 264.7 cells or bone marrow mononuclear cells at concentrations ≤ 40 μM. Luteolin inhibited osteoclast bone resorption with an EC50 of approximately 2.5 μM, without affecting osteoclast actin ring formation. Luteolin-treated osteoclasts produced deeper resorption pits, but with decreased surface area, resulting in overall decreased pit volume. Luteolin did not affect transcription, or protein levels, of V-ATPase subunits a3, d2 and E, or V1V0 assembly. Previous work has shown that luteolin can be effective in reducing bone resorption, and our studies suggest that this effect of luteolin may be through disruption of osteoclast V-ATPase a3–d2 interaction. We conclude that the V-ATPase a3–d2 interaction is a viable target for novel antiresorptive therapeutics that potentially preserve osteoclast–osteoblast signaling important for bone remodeling
Targeted Pten deletion plus p53-R270H mutation in mouse mammary epithelium induces aggressive claudin-low and basal-like breast cancer
No full textBACKGROUND:
Triple-negative breast cancer (TNBC), an aggressive disease comprising several subtypes including basal-like and claudin-low, involves frequent deletions or point mutations in TP53, as well as loss of PTEN. We previously showed that combined deletion of both tumor suppressors in the mouse mammary epithelium invariably induced claudin-low-like TNBC. The effect of p53 mutation plus Pten deletion on mammary tumorigenesis and whether this combination can induce basal-like TNBC in the mouse are unknown.
METHODS:
WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) composite mice were generated in which Pten is deleted and a p53-R270H mutation in the DNA-binding domain is induced upon expression of Cre-recombinase in pregnancy-identified alveolar progenitors. Tumors were characterized by histology, marker analysis, transcriptional profiling [GEO-GSE75989], bioinformatics, high-throughput (HTP) FDA drug screen as well as orthotopic injection to quantify tumor-initiating cells (TICs) and tail vein injection to identify lung metastasis.
RESULTS:
Combined Pten deletion plus induction of p53-R270H mutation accelerated formation of four distinct mammary tumors including poorly differentiated adenocarcinoma (PDA) and spindle/mesenchymal-like lesions. Transplantation assays revealed highest frequency of TICs in PDA and spindle tumors compared with other subtypes. Hierarchical clustering demonstrated that the PDA and spindle tumors grouped closely with human as well as mouse models of basal and claudin-low subtypes, respectively. HTP screens of primary Pten(∆):p53(∆) vs. Pten(∆):p53(R270H) spindle tumor cells with 1120 FDA-approved drugs identified 8-azaguanine as most potent for both tumor types, but found no allele-specific inhibitor. A gene set enrichment analysis revealed increased expression of a metastasis pathway in Pten(∆):p53(R270H) vs. Pten(∆):p53(∆) spindle tumors. Accordingly, following tail vein injection, both Pten(∆):p53(R270H) spindle and PDA tumor cells induced lung metastases and morbidity significantly faster than Pten(∆):p53(∆) double-deletion cells, and this was associated with the ability of Pten(∆):p53(R270H) tumor cells to upregulate E-cadherin expression in lung metastases.
CONCLUSIONS:
Our results demonstrate that WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice represent a tractable model to study basal-like breast cancer because unlike p53 deletion, p53(R270H) mutation in the mouse does not skew tumors toward the claudin-low subtype. The WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice develop basal-like breast cancer that is enriched in TICs, can readily form lung metastasis, and provides a preclinical model to study both basal-like and claudin-low TNBC in immune-competent mice
MicroRNA mediated lysosomal enzymes down-regulation in cell of peripheral system from aAlzheimer's disease patients
No full textAlzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by neurofibrillary tangles, extracellular amyloid plaques and neuroinflammation. New evidences have shown that the lysosomal system might be a crossroad in which etiological factors in AD pathogenesis converge.
This study shows that several lysosomal enzymes, including Cathepsin B, D, S, -Gal, -Man and -Hex, were less expressed in monocytes and lymphocytes from patients with a clinical diagnosis of AD dementia compared to cells from healthy controls.
In vitro experiments of gain and loss of function suggest that down-regulation is a direct consequence of miR-128 up-regulation found in AD related cells. The present study also demonstrates that miR-128 inhibition in monocytes from AD patients improves amyloid β-peptide (Aβ42) degradation.
These results could contribute to clarify the molecular mechanisms which affect the imbalanced amyloid production/clearance involved in the pathogenesis of Alzheimer’s disease
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