Top PDF Cell-Selective Chemoproteomics for Biological Discovery

Cell-Selective Chemoproteomics for Biological Discovery

Cell-Selective Chemoproteomics for Biological Discovery

proteomics for biological discovery.” Curr. Opin. Chem. Biol. 2017, 36, 50-57. DOI: 10.1016/j.cbpa.2016.12.026 1.1 Abstract Proteomic plasticity is a hallmark of development and adaptation. Organisms rely upon translational regulation to respond rapidly to both internal and external cues. Convenience, and in some cases necessity, drove early systems-level studies of translational control to adopt transcript-based proxies instead of direct protein measurements. However, discordance between steady-state transcript and protein levels argues for the development of methods that more accurately quantify expression. Recent advances in cell-specific translatomics and proteomics—fueled in part by the development of bioorthogonal chemistries, more sensitive mass spectrometers and more advanced mining algorithms—
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Cell-selective proteomics for biological discovery

Cell-selective proteomics for biological discovery

Notably, all amino acids and enrichment media needed for BONCAT experiments are commercially available. Stochastic Orthogonal Recoding of Translation (SORT) Chin and coworkers have developed a residue-specific ncAA-labeling technology termed stochastic orthogonal recoding of translation (SORT), which – like BONCAT – allows chemoselective modification and enrichment of newly synthesized cellular proteins. SORT relies on expression of a pyrrolysyl-tRNA synthetase and its cognate tRNA [39,40]. Using this method, Elliott et al. cell-selectively labeled and identified proteins made during different stages of larval growth in Drosophila. Importantly, SORT allows the anticodon of the cognate tRNA to be changed to direct the ncAA to different sets of codons in the labeled proteins. Elliott et al. have characterized the enrichment process and found that tagging at different codons leads to the enrichment of overlapping, but distinct sets of proteins [41]. The authors noted that simultaneous expression of multiple tRNAs (i.e., tRNA-Ala, -Ser and -Met) increases labeling efficiency. Furthermore, Elliott et al. found that enrichment after tagging improves detection of low-abundance proteins.
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Biological effects of selective COX-2 inhibitor NS398 on human glioblastoma cell lines

Biological effects of selective COX-2 inhibitor NS398 on human glioblastoma cell lines

Indeed, studies with this COX-2 inhibitor mostly highlight Fig. 9 NS398 inhibited wound healing ability of GBM cell lines. Representative phase-contrast micrographs and quantification of scratch assays performed in a U87MG and b T98G GBM cell lines treated with NS398 or with drug vehicle DMSO (CNTR), immediately after the scratch at the initial time (T0) and after 8 h and 24 h. Representative phase-contrast micrographs and quantification of scratch assays performed in (c) U87MG and d T98G GBM cell lines after incubation with EV derived from control (CNTR) neurospheres ( + EV CNTR-NS) or NS398-treated neurospheres ( + EV NS398-NS). Original magnification 10x. The results, expressed as % wound closure vs relative T0 (mean ± SD), are representative of two independent experiments in duplicate. For comparative analysis of groups of data, two-way ANOVA followed by Bonferroni post hoc test was used (*p < 0.05,
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Chemoproteomics reveals Toll-like receptor fatty acylation

Chemoproteomics reveals Toll-like receptor fatty acylation

Having made the previous discovery of the critical role of palmitoylation of IFITMs in the innate antiviral immune response [7,14,15], we sought to determine whether any additional IFN-induced proteins are regu- lated by palmitoylation. For our experiments, we chose to use murine antigen presenting cells (DC2.4) and MEFs because these cell lines are responsive to type I IFNs [14], are amenable to labeling with the alk-16 re- porter of protein palmitoylation [6,14] and serve as a control for one another in that type I IFN should induce a similar set of proteins in both cell types. Additionally, this analysis would provide a valuable comparison of the general palmitoylomes of two cell types (myeloid and non-myeloid) with unique functions. DC2.4 cells and MEFs were either left untreated or were treated with IFNα for four hours prior to metabolic labeling with alk-16 in the presence or absence of IFNα for an additional two hours. Cell lysates were reacted with az- rho or az-biotin via click chemistry (Figure 1A). Az-rho- labeled proteins were separated by SDS-PAGE and visualized by fluorescence gel scanning (Figure 1B). Anti-IFITM3 and anti-GAPDH blotting were used as controls for the activity of IFNα on these cells and for loading, respectively. The fluorescent profiles of the two cell types did not indicate that drastic changes to palmi- toylation occur upon IFNα treatment. However, one specific band could be seen uniquely in the IFNα- treated samples, appearing at approximately 15 kDa, po- tentially corresponding to the molecular weight of the IFITMs (Figure 1B).
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Discovery of selective, antimetastatic and anti cancer stem cell metallohelices via post assembly modification

Discovery of selective, antimetastatic and anti cancer stem cell metallohelices via post assembly modification

Introduction Lehn envisaged in the original report 1 that helicates 2–4 – self- assembling multimetallic coordination compounds – may nd uses in biochemistry. Indeed, while their underlying chemistry is very different to that of the small cationic a-helical peptide units that are deployed in nature for e.g. signalling, structural and host-defence roles, 5 – 7 some such metallofoldamers 8 have similar dimensions and charge. With this in mind we have developed several classes of water-compatible, optically pure metallohelix compounds, 9–11 each of which has unique proper- ties, including a growing list of peptide-like behaviours: binding of DNA motifs, 12 anticancer activity, 5,11,13 and the inhibition of e.g. amyloid-b aggregation, 12,14 enzyme activity 15,16 and ice recrystallization. 17 Thus, while we cannot expressly mimic the exquisite architectures of natural peptides, we are motivated to seek methods by which diverse metallohelices might be rapidly accessed and new biological properties discovered and optimised. 18
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Discovery of orbital selective Cooper pairing in FeSe

Discovery of orbital selective Cooper pairing in FeSe

20 Figure 1 Bogoliubov Quasiparticle Interference Model for FeSe (A) Top view of FeSe crystal structure. Dashed lines represent the 1-Fe unit cell, and the actual unit cell is shown using solid lines. The unit cell of FeSe is distorted in the nematic phase with a Fe >b Fe . Throughout this paper we define the 𝑥-axis, 𝑘 ⃗ 𝑥 -axis and 𝑞 𝑥 -axis to all be parallel to a Fe -axis, so that labels of orbitals like d xy or d yz or 𝑘 ⃗ -space locations and states, are equally valid in both nematic domains.

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The discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition

The discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition

Although BRD9 has been reported as a component of the chromatin remodeling SWI/SNF BAF complex, 41−43 further information regarding its role within disease is somewhat limited. 44 As such, a chemical probe for the BRD9 bromodomain would be invaluable in better understanding the biological role and therapeutic potential of this reader module. The Structural Genomics Consortium have disclosed bromosporine (15), 45 a broad spectrum bromodomain inhibitor, which shows a thermal shift of +3.9 °C at BRD9, suggesting bromodomain binding. In addition, structurally similar compound 16 was reported as the fi rst nonselective inhibitor of BRD9. 46,47 This compound shows mixed bromodomain pharmacology, with submicromolar activity against BRD9, BRD4, CECR2, and CREBBP. To the best of our knowledge, there are no BRD9 selective chemical probes described in the literature to date. 48 Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for BRD9. The development of I-BRD9 was driven by iterative medicinal chemistry, utilizing structure based design to deliver nanomolar potency at BRD9, >700-fold selectivity over the BET family and >70-fold against a panel of 34 bromodomains. I-BRD9 meets and exceeds the chemical probe criteria we defined at the outset of this project, inspired by literature from Bunnage and co- workers, 49 who highlighted the importance of high quality chemical probes for target validation: 100 nM or greater potency against the bromodomain of BRD9 as determined by a biochemical assay; 100-fold selectivity over the BET family of bromodomains; 30-fold selectivity over other bromodomain families; cellular activity; and broader selectivity over a range of receptor sites, ion channels, transporters, and enzymes.
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The discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition

The discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition

Although BRD9 has been reported as a component of the chromatin remodeling SWI/SNF BAF complex, 41−43 further information regarding its role within disease is somewhat limited. 44 As such, a chemical probe for the BRD9 bromodomain would be invaluable in better understanding the biological role and therapeutic potential of this reader module. The Structural Genomics Consortium have disclosed bromosporine (15), 45 a broad spectrum bromodomain inhibitor, which shows a thermal shift of +3.9 ° C at BRD9, suggesting bromodomain binding. In addition, structurally similar compound 16 was reported as the fi rst nonselective inhibitor of BRD9. 46,47 This compound shows mixed bromodomain pharmacology, with submicromolar activity against BRD9, BRD4, CECR2, and CREBBP. To the best of our knowledge, there are no BRD9 selective chemical probes described in the literature to date. 48 Herein, we report the discovery of I-BRD9, the fi rst selective cellular chemical probe for BRD9. The development of I-BRD9 was driven by iterative medicinal chemistry, utilizing structure based design to deliver nanomolar potency at BRD9, >700-fold selectivity over the BET family and >70-fold against a panel of 34 bromodomains. I-BRD9 meets and exceeds the chemical probe criteria we de fi ned at the outset of this project, inspired by literature from Bunnage and co- workers, 49 who highlighted the importance of high quality chemical probes for target validation: 100 nM or greater potency against the bromodomain of BRD9 as determined by a biochemical assay; 100-fold selectivity over the BET family of bromodomains; 30-fold selectivity over other bromodomain families; cellular activity; and broader selectivity over a range of receptor sites, ion channels, transporters, and enzymes.
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Knowledge discovery for stochastic models of biological systems

Knowledge discovery for stochastic models of biological systems

For both approaches we coupled statistical methods with high performance parallel algorithms in order to properly face with the computational de- mands of stochastic simulations. In order to evaluate the proposed approaches and methodologies we con- sidered several case studies. Every method is presented and consequently explained and tested on a particular problem. The evolutionary inference framework has been tested on a well known benchmarking problem, the thermal isomerization of α-pinene. Analysis of model properties has been tested on a cell cycle model, while the approach to identify and estimate the parameter effects on a desired model output has been tested on a predator- prey model to evaluate the relationship among parameters and oscillation frequencies.
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Selective Green Device Discovery for Device-to-Device Communication

Selective Green Device Discovery for Device-to-Device Communication

Copyright © 2017 Universitas Ahmad Dahlan. All rights reserved. 1. Introduction Due to the explosion of the mobile service and user demand [1], the mobile network is expected to be highly congested in the near future [2]. To cope with this problem, device-to- device (D2D) communication offers direct communication between devices in proximity, allowing the user plane traffic to be exchanged, without burdening the network [3]. Furthermore, the D2D communication, together with Internet of Things (IoT) and small cell access is also proposed as an upcoming architectural revolution in 5G communication [3]. Therefore, D2D communication will play a major role in the next generation wireless network.
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MicroRNA in Oral Squamous Cell Carcinoma

and Oral Potentially Malignant Lesions:

from biological discovery to clinical utility.

MicroRNA in Oral Squamous Cell Carcinoma and Oral Potentially Malignant Lesions: from biological discovery to clinical utility.

5. Discussion Histological diagnosis cannot evaluate the risk of malignant transformation. Thus, miRNA has revealed to be good biomarkers for the management of these lesions. It seems that miRNAs can regulate important genetic steps that lead a dysplastic cell to transform into a cancer cell. In the present study, we explore the expression of miRNA in the malignant transformation of OPML and in aggressive OSCC cells and tumor samples.

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Innovation of System Biological Approach in Computational Drug Discovery

Innovation of System Biological Approach in Computational Drug Discovery

validating selected compounds and targets (19). Highly reproducible or even automated approaches to cell biology, however, seem more likely to contribute to the large-scale compound and gene function analyses desired by industry and required as a basis for modeling efforts. Assays are generally designed to isolate individual pathways and to minimize biological complexity. This ‘systematic biology’

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Stem cell biology and drug discovery

Stem cell biology and drug discovery

identified have typically replaced one or more of the reprogramming factors or have improved the efficiency of the overall process. Many of the screens have provided some insight into the mechanism of reprogramming. One example of such a screen was based on a simple experiment designed to identify a small molecule capable of replacing the transcription factor Sox2 [18]. Mouse embryo fibroblasts were transduced with retroviruses coding for Klf­4, Oct­4 and c­Myc, but not Sox2. Under those conditions, no true iPSC colonies formed. The cells were then treated with agents selected from an annotated compound library enriched in small molecules that modulate intracellular signaling. The most potent hit was an inhibitor of transforming growth factor (TGF)­β signal ing. The surprise was in the way it acted: it increased expression of Nanog, another transcription factor with reprogramming activity. Furthermore, it affected not the starting cell population, but a population of partially reprogrammed intermediate cells that appeared 1 to 2 weeks after virus addition. This work, along with many other reports, demonstrates that reprogramming can be achieved in several, perhaps numerous, ways, with cells traversing different paths of dedifferentiation via many transient states of partial dedifferentiation. These repro­ gram ming intermediates are, in a sense, artificial, being created as a result of an artificial process. This concept will be explored later in the context of regulating a real biological process: cell differentiation.
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Discovery of functionally selective C5aR2 ligands: novel modulators of C5a signalling.

Discovery of functionally selective C5aR2 ligands: novel modulators of C5a signalling.

In summary, here we report the discovery of two functionally selective ligands (P32, P59) for C5aR2, which are partial agonists (when compared to C5a) for β-arrestin 2 recruitment. Importantly, our current and previous 21 data demonstrate that P32 and P59 are devoid of activity (either as agonists or antagonists) when screened against C3aR and C5aR1 in a range of assays, and can hence be considered as functionally selective ligands for C5aR2. We acknowledge that the utility of these ligands will be as tool compounds, rather than direct therapeutic candidates, due to their low affinity for C5aR2. Nevertheless these tool ligands provide a good starting point to investigate the biological role of C5aR2. This is verified by our demonstration that P32 could inhibit C5a-induced neutrophil mobilization in mice, in a C5aR2- dependent manner. The discovery of these functionally selective C5aR2 ligands will therefore now allow complement researchers to selectively probe the functional role of C5aR2 in vitro and in vivo. We plan to use these ligands as a medicinal chemistry template for development of higher affinity selective ligands for C5aR2. Indeed, the ability of P32 to inhibit C5a-mediated ERK1/2 activation and selectively inhibit IL-6 secretion from human macrophages, and block C5a-mediated neutrophil mobilization in vivo, indicates that potent and selective C5aR2 agonists may have therapeutic potential as treatments for inflammatory disease.
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Integration of Genomic Data Enables Selective Discovery of Breast Cancer Drivers

Integration of Genomic Data Enables Selective Discovery of Breast Cancer Drivers

drivers can impact more patients ( Figure 1 A). However, to date, this possibility has been limited by the difficultly of distinguishing passengers and drivers in the majority of SCNAs. Here, we have presented a major advance in addressing this challenge, using a method that integrates data from primary tu- mors with functional assays on cell lines to prioritize candidate drivers. The unparalleled sensitivity and specificity of Helios enabled us to execute the first reported systematic validation of an algorithm designed to identify driver genes. Helios’s perfor- mance was confirmed by a success rate of 10/12 candidates in an anchorage-independent growth assay, successfully charac- terizing several regions for which there was no previously impli- cated driver. Importantly, because we selected the genes for validation based on their amplification significance (ISAR score), rather than their Helios score, we expect that this success rate will extend to additional regions that have equally strong Helios scores. Moreover, many of these genes are amplified in addi- tional epithelial cancers (e.g., C6orf203, NIT1, ZNF652) suggest- ing possible drivers in those cancers as well.
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Discovery of biological networks from diverse functional genomic data

Discovery of biological networks from diverse functional genomic data

Although the cross-talk across all of these biological processes has not yet been well characterized, evidence in the literature supports these predicted connections. For instance, the expression pattern of CBF1, INO2, or SWI5 is well correlated with the expression of NOP7 (for example, as cells undergo diauxic shift and during sporulation, CBF1 and NOP7 are co-expressed with a Pearson correlation of greater than 0.8 [33-35]). Du and Stillman [36] found that Nop7/Yph1, a protein required for the biogenesis of 60S ribosomal subunits [37-39], associates with the origin recog- nition complex, cell cycle-related proteins, and MCM pro- teins. As cells are depleted of Nop7p, they exhibit cell cycle arrest, and in wild-type cells, Nop7 levels vary in response to different carbon sources [39]. Taken together, these previous experimental results support our prediction linking meta- bolic pathways, the cell cycle, and ribosome assembly. It is important to note that while the characterization of Nop7 is consistent with this prediction, the individual experiments with Nop7 described above were not part of the input data to our system. Rather, our system was able to make the pre- dicted links across these functional groups based on other heterogeneous, and mostly high throughout, data through bioPIXIE integration and network analysis. Thus, cross-talk analysis using bioPIXIE is effective in identifying novel A map of cross-talk between 363 biological groups in S. cerevisiae
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Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

expression and image-based profiles for over 12,000 bioactive compounds and over 17,000 diversity-oriented synthesis (DOS)-based molecules for which HTS results from up to 178 cell-based screens were available (Wawer et al., 2014b). We asked if the profiling data could select for compounds with diverse bioactivities, as judged by comparing patterns of activity in cells or against target proteins. Compounds selected based on varied activity in the imaging and gene expression assay profiles significantly outperformed compounds selected randomly or based on chemical diversity when their performance diversity in historical HTS assays was compared. In addition, we discovered thousands of compounds, many of which formed scaffold-based clusters, that induced cell morphology and gene expression patterns not seen within the 12,000 bioactives, likely hinting at novel mechanisms of action. These methods and others that yield multiplexed data reporting
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Chemical Genomics: A Systematic Approach in Biological Research and Drug Discovery

Chemical Genomics: A Systematic Approach in Biological Research and Drug Discovery

mRNAs are prepared from the treated and untreated cell or organism, and then used to produce fluorescence- labeled cDNAs to hybridize DNA microarrays and generate gene expression profiles. By comparing the differences between the profiles before and after drug treatment, genes whose expression is modulated by the chemical ligands are then identified. These genes are further classified into pools based on similar functions or co-regulation in the cell. This information can often reveal specific transcription factors and other regulators for each gene pool, thereby allowing assembly of potential regulatory pathways involving the drug target. A nice example is provided by a recent study of yeast histone deacetylases (HDACs) (Bernstein et al. 2000). In that study, three yeast HDACs, HDA1, RPD3 and SIR2, were examined using the drug trichostatin A (TSA) and deletion of the genes. Hda1 and Rpd3, but not Sir2, are sensitive to TSA. The authors elegantly demonstrated that individual HDACs have specific roles in distinct transcriptional pathways as well as some limited overlapping functions. It is now realized that genes are regulated as networks. Many genes are co-regulated in response to unique cellular conditions.
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Induced cancer stem-like cells as a model for biological screening and discovery of agents targeting phenotypic traits of cancer stem cell

Induced cancer stem-like cells as a model for biological screening and discovery of agents targeting phenotypic traits of cancer stem cell

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Cancer stem cells (CSCs) retain the capacity to propagate themselves through self-renewal and to produce heterogeneous lineages of cancer cells constituting the tumor. Novel drugs that target CSCs can potentially eliminate the tumor initiating cell population therefore resulting in complete cure of the cancer. We recently established a CSC-like model using induced pluripotent stem cell (iPSC) technology to reprogram and partially differentiate human mammary epithelial MCF-10A cells. Using the induced CSC-like (iCSCL) model, we developed a phenotypic drug assay system to identify agents that inhibit the stemness and self-renewal properties of CSCs. The selectivity of the agents was assessed using three distinct assays characterized by cell viability, cellular stemness and tumor sphere formation. Using this approach, we found that withaferin A (WA), an Ayurvedic medicine constituent, was a potent inhibitor of CSC stemness leading to cellular senescence primarily via the induction of p21 Cip1 expression. Moreover, WA exhibited strong anti-tumorigenic activity against the iCSCL. These results indicate that our iCSCL model provides an innovative high throughput platform for a simple, easy, and cost-effective method to search for novel CSC-targeting drugs. Furthermore, our current study identified WA as a putative drug candidate for abrogating the stemness and tumor initiating ability of CSCs.
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Induced cancer stem-like cells as a model for biological screening and discovery of agents targeting phenotypic traits of cancer stem cell

Induced cancer stem-like cells as a model for biological screening and discovery of agents targeting phenotypic traits of cancer stem cell

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Cancer stem cells (CSCs) retain the capacity to propagate themselves through self-renewal and to produce heterogeneous lineages of cancer cells constituting the tumor. Novel drugs that target CSCs can potentially eliminate the tumor initiating cell population therefore resulting in complete cure of the cancer. We recently established a CSC-like model using induced pluripotent stem cell (iPSC) technology to reprogram and partially differentiate human mammary epithelial MCF-10A cells. Using the induced CSC-like (iCSCL) model, we developed a phenotypic drug assay system to identify agents that inhibit the stemness and self-renewal properties of CSCs. The selectivity of the agents was assessed using three distinct assays characterized by cell viability, cellular stemness and tumor sphere formation. Using this approach, we found that withaferin A (WA), an Ayurvedic medicine constituent, was a potent inhibitor of CSC stemness leading to cellular senescence primarily via the induction of p21 Cip1 expression. Moreover, WA exhibited strong anti-tumorigenic activity against the iCSCL. These results indicate that our iCSCL model provides an innovative high throughput platform for a simple, easy, and cost-effective method to search for novel CSC-targeting drugs. Furthermore, our current study identified WA as a putative drug candidate for abrogating the stemness and tumor initiating ability of CSCs.
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