Frenzel A, Zirath H, Vita M, Albihn A, Henriksson MA. "PLoS One. 2011"
Expression of MYC is deregulated inside a wide range of human cancers, and it is normally associated with aggressive illness and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would therefore be advantageous for that therapy of those tumors.
For this goal we used cell lines with conditional MYCN or c-MYC expression, to screen a library of eighty standard cytotoxic compounds for their ability to minimize tumor cell viability and/or development in a MYC dependent way.
We discovered that 25% from the researched compounds induced apoptosis and/or inhibited proliferation in a MYC-specific method. The activities of the majority of those had been improved both by c-MYC or MYCN over-expression.
Interestingly, these compounds were acting on unique mobile targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine) and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide) also as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C).
Our data show that MYC overexpression sensitizes cells to disruption of precise pathways and that in most cases c-MYC and MYCN overexpression have similar results within the responses to cytotoxic compounds.
Treatment from the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, when doxorubicin and also the small molecule MYRA-A was located to disrupt MYC-Max interaction.
We conclude the MYC pathway is simply specific by a subset of standard cytotoxic medications at the moment made use of inside the clinic. Elucidating the mechanisms underlying their specificity in direction of MYC might be of value for optimizing remedy of tumors with MYC deregulation.
Our data also underscores that MYC is an desirable target for novel therapies and that mobile screenings of Compound Libraries is usually a strong tool for figuring out compounds with a desired biological activity.
2012年1月4日星期三
Polymers for siRNA Delivery
Wagner E. "Acc Chem Res. 2011 Dec"
Synthetic small interfering RNA (siRNA). offers an exciting novel health-related chance. Though scientists consent that siRNA could have an awesome therapeutic impact, the needed extracellular and intracellular delivery of these molecules in to the disease-associated target cells offers the main roadblock for the broader translation of these molecules into medicines. Therefore, the style of adequate delivery technologies has utmost importance.
Viruses are natural masterpieces of nucleic acid delivery and current chemists and drug delivery specialists having a template for your design of synthetic carriers for synthetic nucleic acids for instance siRNA. They've been developed into gene vectors and have provided convincing successes in gene treatment.
Optimized by biological evolution, viruses are programmed to be dynamic and bioresponsive as they enter residing cells, and they carry out their features in a precisely defined sequence. However, since they are synthesized inside living cells and with in a natural way offered nucleotides and amino acids, the chemistry of viruses is limited.
With the use of diverse synthetic molecules and macromolecules, chemists can provide delivery remedies past the scope in the natural evolution of viruses. This Account describes the style and synthesis of "synthetic siRNA viruses."
These structures contain components that mimic the delivery features of viral particles and surface area domains that shield in opposition to undesired biological interactions and allow specific host cell receptor binding through the presentation of a number of concentrating on ligands. For example, cationic polymers can reversibly bundle one or a lot more siRNA molecules into nanoparticle cores to guard them against a degradative bioenvironment.
After internalization by receptor-mediated endocytosis to the acidifying endosomes of cells, artificial siRNA can escape from these vesicles by way of the activation of membrane-disruption domains as viruses do and attain the cytoplasm, the location of RNA interference. This multistep process presents an attractive problem for chemists.
Similar for the design of prodrugs, the functional domains of these methods need to be activated within a dynamic mode, triggered by conformational adjustments or bond cleavages in the related microenvironment such as the acidic endosome or disulfide-reducing cytoplasm. These chemical analogues of viral domains are usually synthetically easier and a lot more very easily available molecules than viral proteins.
Their exact assembly into multifunctional macromolecular and supramolecular structures is facilitated by enhanced analytical tactics, precise orthogonal conjugation chemistries, and sequence-defined polymer syntheses.
The chemical evolution of microdomains working with chemical libraries. and macromolecular and supramolecular evolution could give crucial strategies for optimizing siRNA carriers to chosen medical indications.
Synthetic small interfering RNA (siRNA). offers an exciting novel health-related chance. Though scientists consent that siRNA could have an awesome therapeutic impact, the needed extracellular and intracellular delivery of these molecules in to the disease-associated target cells offers the main roadblock for the broader translation of these molecules into medicines. Therefore, the style of adequate delivery technologies has utmost importance.
Viruses are natural masterpieces of nucleic acid delivery and current chemists and drug delivery specialists having a template for your design of synthetic carriers for synthetic nucleic acids for instance siRNA. They've been developed into gene vectors and have provided convincing successes in gene treatment.
Optimized by biological evolution, viruses are programmed to be dynamic and bioresponsive as they enter residing cells, and they carry out their features in a precisely defined sequence. However, since they are synthesized inside living cells and with in a natural way offered nucleotides and amino acids, the chemistry of viruses is limited.
With the use of diverse synthetic molecules and macromolecules, chemists can provide delivery remedies past the scope in the natural evolution of viruses. This Account describes the style and synthesis of "synthetic siRNA viruses."
These structures contain components that mimic the delivery features of viral particles and surface area domains that shield in opposition to undesired biological interactions and allow specific host cell receptor binding through the presentation of a number of concentrating on ligands. For example, cationic polymers can reversibly bundle one or a lot more siRNA molecules into nanoparticle cores to guard them against a degradative bioenvironment.
After internalization by receptor-mediated endocytosis to the acidifying endosomes of cells, artificial siRNA can escape from these vesicles by way of the activation of membrane-disruption domains as viruses do and attain the cytoplasm, the location of RNA interference. This multistep process presents an attractive problem for chemists.
Similar for the design of prodrugs, the functional domains of these methods need to be activated within a dynamic mode, triggered by conformational adjustments or bond cleavages in the related microenvironment such as the acidic endosome or disulfide-reducing cytoplasm. These chemical analogues of viral domains are usually synthetically easier and a lot more very easily available molecules than viral proteins.
Their exact assembly into multifunctional macromolecular and supramolecular structures is facilitated by enhanced analytical tactics, precise orthogonal conjugation chemistries, and sequence-defined polymer syntheses.
The chemical evolution of microdomains working with chemical libraries. and macromolecular and supramolecular evolution could give crucial strategies for optimizing siRNA carriers to chosen medical indications.
2011年12月30日星期五
High-Throughput Fluorescence Polarization Assay for Chemical Library Screening
Zhai D, Godoi P, Sergienko E, Dahl R, Chan X, Brown B, Rascon J, Hurder A, Su Y, Chung TD, Jin C, Diaz P, Reed JC. "J Biomol Screen. 2011 Dec"
Overexpression with the anti-apoptotic Bcl-2 loved ones proteins occurs normally in human cancers. Bfl-1 is highly expressed in a few kinds of malignant cells, contributing considerably to tumor mobile survival and chemoresistance. Hence, it would be appealing to possess chemical antagonists of Bfl-1.
To this end, we devised a fluorescence polarization assay (FPA) applying Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was utilized for high-throughput screening of chemical library.
Approximately 66 000 compounds had been screened for your potential to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ?Y50% displacement. Immediately after dose-response analysis and confirmation working with a secondary assay according to time-resolved fluorescence resonance power transfer (TR-FRET), two teams of Bfl-1-specific inhibitors were identified, which includes chloromaleimide and sulfonylpyrimidine sequence compounds.
Fluorescence Polarization Assay (FPA) generated for each from the 6 anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs in the sulfonylpyrimidine collection had been synthesized and in contrast with the original strike for Bfl-1 binding by each FPAs and TR-FRET assays.
The ensuing structure-activity relation analysis led for the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of utilizing the HTS assay for discovery of selective chemical inhibitors of Bfl-1.
Overexpression with the anti-apoptotic Bcl-2 loved ones proteins occurs normally in human cancers. Bfl-1 is highly expressed in a few kinds of malignant cells, contributing considerably to tumor mobile survival and chemoresistance. Hence, it would be appealing to possess chemical antagonists of Bfl-1.
To this end, we devised a fluorescence polarization assay (FPA) applying Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was utilized for high-throughput screening of chemical library.
Approximately 66 000 compounds had been screened for your potential to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ?Y50% displacement. Immediately after dose-response analysis and confirmation working with a secondary assay according to time-resolved fluorescence resonance power transfer (TR-FRET), two teams of Bfl-1-specific inhibitors were identified, which includes chloromaleimide and sulfonylpyrimidine sequence compounds.
Fluorescence Polarization Assay (FPA) generated for each from the 6 anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs in the sulfonylpyrimidine collection had been synthesized and in contrast with the original strike for Bfl-1 binding by each FPAs and TR-FRET assays.
The ensuing structure-activity relation analysis led for the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of utilizing the HTS assay for discovery of selective chemical inhibitors of Bfl-1.
The monoamine oxidase A, a broad-spectrum inhibitor of fungal ABC and MFS transporter.
Holmes AR, Keniya MV, Ivnitski-Steele I, Monk BC, Lamping E, Sklar LA, Cannon RD. "Antimicrob Agents Chemother. 2011 Dec"
Resistance to the commonly employed azole antifungal fluconazole (FLC) can develop on account of over-expression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to conquering this resistance would be to identify inhibitors of those efflux pumps.
We have created a pump assay appropriate for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyper-expressing person transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess higher resistance to azoles and also other pump substrates as opposed to parental host strain.
A flow cytometry-based HTS, which measured elevated intracellular retention with the luminescent pump substrate rhodamine 6G (R6G) inside yeast cells, was utilized to screen the Prestwick Chemical Library (PCL) of 1200 marketed medication. 9 compounds were determined as hits as well as the monoamine-oxidase A inhibitor (MAOI) clorgyline was recognized being an inhibitor of two Do. albicans ABC efflux pumps, CaCdr1p and CaCdr2p.
Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC-resistance of S. cerevisiae strains expressing other person fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the Do. albicans major facilitator superfamily (MFS) transporter Mdr1p. Recombinant strains had been also chemosensitized by clorgyline to other azoles (itraconazole and miconazole (MCZ)).
Importantly, clorgyline confirmed synergy with FLC towards FLC-resistant Do. albicans clinical isolates and also a Do. glabrata strain and inhibited R6G efflux from a FLC-resistant Do. albicans clinical isolate. Clorgyline is usually a novel broad-spectrum inhibitor of two courses of fungal efflux pumps that functions synergistically with azoles versus azole-resistant C. albicans and Do. glabrata strains.
Resistance to the commonly employed azole antifungal fluconazole (FLC) can develop on account of over-expression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to conquering this resistance would be to identify inhibitors of those efflux pumps.
We have created a pump assay appropriate for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyper-expressing person transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess higher resistance to azoles and also other pump substrates as opposed to parental host strain.
A flow cytometry-based HTS, which measured elevated intracellular retention with the luminescent pump substrate rhodamine 6G (R6G) inside yeast cells, was utilized to screen the Prestwick Chemical Library (PCL) of 1200 marketed medication. 9 compounds were determined as hits as well as the monoamine-oxidase A inhibitor (MAOI) clorgyline was recognized being an inhibitor of two Do. albicans ABC efflux pumps, CaCdr1p and CaCdr2p.
Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC-resistance of S. cerevisiae strains expressing other person fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the Do. albicans major facilitator superfamily (MFS) transporter Mdr1p. Recombinant strains had been also chemosensitized by clorgyline to other azoles (itraconazole and miconazole (MCZ)).
Importantly, clorgyline confirmed synergy with FLC towards FLC-resistant Do. albicans clinical isolates and also a Do. glabrata strain and inhibited R6G efflux from a FLC-resistant Do. albicans clinical isolate. Clorgyline is usually a novel broad-spectrum inhibitor of two courses of fungal efflux pumps that functions synergistically with azoles versus azole-resistant C. albicans and Do. glabrata strains.
2011年12月21日星期三
Diversity Quantification of Chemical Libraries
Colliandre L, Le Guilloux V, Bourg S, Morin-Allory L. "J Chem Inf Model. 2011 Dec"
High Throughput Screening (HTS) can be a standard technique widely useful to find hit compounds within drug discovery projects. The high costs associated with such experiments have highlighted the necessity to carefully design screening libraries to avoid wasting resources. Molecular diversity is an established concept that's used to this end for many years.
A new approach to help quantify the molecular diversity of screening libraries is usually presented nowdays. The approach is dependent on the Delimited Reference Chemical Subspace (DRCS) strategy, a new method that can be used to delimit the densest subspace spanned by a reference library in a lower life expectancy 2D continuous space. An overall of twenty-two diversity indices have been implemented or adapted to the current methodology, which is used here to take out outliers and obtain another cell-based partition of that subspace.
The behavior these indices was assessed and compared in various extreme situations, and with respect to a set of theoretical rules that a diversity function should satisfy when libraries of different sizes have to be compared. Some gold standard indices are found inappropriate ordinary context, while none of the tested indices behave perfectly in all cases. Five DRCS-based indices accounting for different aspects of diversity were lastly selected, and a simple framework is proposed to use them effectively.
Current drug-discovery strategies are typically 'target-centric' and are based upon high-throughput screening of significant chemical libraries against nominated targets and a selection of lead compounds with optimized 'on-target' capacity and selectivity profiles.
However, high attrition of targeted agents in clinical development claim that combinations of targeted agents is going to be most effective in treating solid tumors in the event the biological networks that allow cancer cells to subvert monotherapies are identified and retargeted. Conventional drug-discovery and advancement strategies are suboptimal for any rational design and advancement of novel drug combinations.
In this article, we highlight several emerging technologies supporting some sort of less reductionist, more agile, drug-discovery and development approach for any rational design, validation, prioritization together with clinical development of innovative drug combinations.
High Throughput Screening (HTS) can be a standard technique widely useful to find hit compounds within drug discovery projects. The high costs associated with such experiments have highlighted the necessity to carefully design screening libraries to avoid wasting resources. Molecular diversity is an established concept that's used to this end for many years.
A new approach to help quantify the molecular diversity of screening libraries is usually presented nowdays. The approach is dependent on the Delimited Reference Chemical Subspace (DRCS) strategy, a new method that can be used to delimit the densest subspace spanned by a reference library in a lower life expectancy 2D continuous space. An overall of twenty-two diversity indices have been implemented or adapted to the current methodology, which is used here to take out outliers and obtain another cell-based partition of that subspace.
The behavior these indices was assessed and compared in various extreme situations, and with respect to a set of theoretical rules that a diversity function should satisfy when libraries of different sizes have to be compared. Some gold standard indices are found inappropriate ordinary context, while none of the tested indices behave perfectly in all cases. Five DRCS-based indices accounting for different aspects of diversity were lastly selected, and a simple framework is proposed to use them effectively.
Current drug-discovery strategies are typically 'target-centric' and are based upon high-throughput screening of significant chemical libraries against nominated targets and a selection of lead compounds with optimized 'on-target' capacity and selectivity profiles.
However, high attrition of targeted agents in clinical development claim that combinations of targeted agents is going to be most effective in treating solid tumors in the event the biological networks that allow cancer cells to subvert monotherapies are identified and retargeted. Conventional drug-discovery and advancement strategies are suboptimal for any rational design and advancement of novel drug combinations.
In this article, we highlight several emerging technologies supporting some sort of less reductionist, more agile, drug-discovery and development approach for any rational design, validation, prioritization together with clinical development of innovative drug combinations.
2011年12月20日星期二
Physical Metagenomic Libraries
Neufeld JD, Engel K, Cheng J, Moreno-Hagelsieb G, Rose DR, Charles TC. "Stand Genomic Sci. 2011 Nov"
Both sequence-based and activity-based exploitation of environmental DNA have supplied unprecedented accessibility for the genomic content of cultivated and uncultivated microorganisms. Despite the fact that scientists deposit microbial strains in tradition collections and DNA sequences in databases, activity-based metagenomic studies typically only publish sequences in the hits retrieved from specific screens.
Physical metagenomic libraries, conceptually similar to whole sequence datasets, are typically not straightforward to get by interested parties subsequent to publication.
In purchase to facilitate unrestricted distribution of metagenomic libraries, we suggest the adoption of open up source metagenomics, in line with the trend towards open up entry publishing, and related to culture- and mutant-strain collections that have been the backbone of classic microbiology and microbial genetics.
The concept of open up source metagenomics includes preparing of physical DNA libraries, ideally in adaptable vectors that facilitate screening inside a variety of host organisms, and pooling of clones so that simple aliquots that contains full libraries may be readily distributed upon ask for. Database deposition of related metadata and sequence data for each library gives scientists with info to choose essentially the most suitable libraries for further research projects.
As a beginning level, we have proven the Canadian MetaMicroBiome Library (CM(2)BL). The CM(two)BL can be a publicly accessible assortment of cosmid libraries containing environmental DNA from soils collected from across Canada, spanning several biomes. The screening libraries had been produced such which the cloned DNA might be simply transferred to Gateway compliant vectors, facilitating purposeful screening in virtually any surrogate microbial host for which you'll find accessible plasmid vectors.
The libraries, which we are placing within the manifeste domain, will be distributed upon ask for with no restriction to members of both the educational study community and market. This article invites the scientific community to adopt this philosophy of open up resource metagenomics to increase the utility of functional metagenomics beyond first publication, circumventing the will need to start from scratch with each new study venture.
Both sequence-based and activity-based exploitation of environmental DNA have supplied unprecedented accessibility for the genomic content of cultivated and uncultivated microorganisms. Despite the fact that scientists deposit microbial strains in tradition collections and DNA sequences in databases, activity-based metagenomic studies typically only publish sequences in the hits retrieved from specific screens.
Physical metagenomic libraries, conceptually similar to whole sequence datasets, are typically not straightforward to get by interested parties subsequent to publication.
In purchase to facilitate unrestricted distribution of metagenomic libraries, we suggest the adoption of open up source metagenomics, in line with the trend towards open up entry publishing, and related to culture- and mutant-strain collections that have been the backbone of classic microbiology and microbial genetics.
The concept of open up source metagenomics includes preparing of physical DNA libraries, ideally in adaptable vectors that facilitate screening inside a variety of host organisms, and pooling of clones so that simple aliquots that contains full libraries may be readily distributed upon ask for. Database deposition of related metadata and sequence data for each library gives scientists with info to choose essentially the most suitable libraries for further research projects.
As a beginning level, we have proven the Canadian MetaMicroBiome Library (CM(2)BL). The CM(two)BL can be a publicly accessible assortment of cosmid libraries containing environmental DNA from soils collected from across Canada, spanning several biomes. The screening libraries had been produced such which the cloned DNA might be simply transferred to Gateway compliant vectors, facilitating purposeful screening in virtually any surrogate microbial host for which you'll find accessible plasmid vectors.
The libraries, which we are placing within the manifeste domain, will be distributed upon ask for with no restriction to members of both the educational study community and market. This article invites the scientific community to adopt this philosophy of open up resource metagenomics to increase the utility of functional metagenomics beyond first publication, circumventing the will need to start from scratch with each new study venture.
2011年12月19日星期一
Discovery of Enoyl-Reductase (FabI) Inhibitors by Molecular Shape and Electrostatic Matching.
Hevener KE, Mehboob S, Su PC, Truong K, Boci T, Deng J, Ghassemi M, Cook JL, Johnson ME. "J Med Chem. 2011 Dec"
Enoyl-acyl carrier protein (ACP) reductase, FabI, is really a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is definitely an NADH-dependent oxidoreductase that functions to cut down enoyl-ACP substrates inside a last stage of the pathway. The absence of this enzyme in humans tends to make it an attractive target for the development of new antibacterial agents.
FabI is known to be unresponsive to structure-based design efforts resulting from a high diploma of induced fit plus a cell versatile loop encompassing the energetic web page.
Here we discuss the improvement, validation, and careful app of a ligand-based virtual display used for your identification of novel inhibitors in the Francisella tularensis FabI focus on.
In this research, 4 known courses of FabI inhibitors had been utilised as templates for virtual screens that concerned molecular form and electrostatic matching. The plan ROCS was made use of to go looking a high throughput screening library for compounds that matched any with the four molecular form queries. Matching compounds were additional refined applying the system EON, which compares and scores compounds by matching electrostatic homes. Employing these approaches, fifty compounds had been chosen, ordered, and examined.
The examined compounds possessed novel chemical scaffolds when compared to the input question compounds. Numerous hits with low micromolar exercise were discovered and follow-up scaffold-based searches resulted within the identification of the lead sequence with submicromolar enzyme inhibition, high ligand efficiency, and also a novel scaffold.
Additionally, just about the most active compounds showed promising whole-cell antibacterial activity from various Gram-positive and Gram-negative species, such as the goal pathogen.
Enoyl-acyl carrier protein (ACP) reductase, FabI, is really a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is definitely an NADH-dependent oxidoreductase that functions to cut down enoyl-ACP substrates inside a last stage of the pathway. The absence of this enzyme in humans tends to make it an attractive target for the development of new antibacterial agents.
FabI is known to be unresponsive to structure-based design efforts resulting from a high diploma of induced fit plus a cell versatile loop encompassing the energetic web page.
Here we discuss the improvement, validation, and careful app of a ligand-based virtual display used for your identification of novel inhibitors in the Francisella tularensis FabI focus on.
In this research, 4 known courses of FabI inhibitors had been utilised as templates for virtual screens that concerned molecular form and electrostatic matching. The plan ROCS was made use of to go looking a high throughput screening library for compounds that matched any with the four molecular form queries. Matching compounds were additional refined applying the system EON, which compares and scores compounds by matching electrostatic homes. Employing these approaches, fifty compounds had been chosen, ordered, and examined.
The examined compounds possessed novel chemical scaffolds when compared to the input question compounds. Numerous hits with low micromolar exercise were discovered and follow-up scaffold-based searches resulted within the identification of the lead sequence with submicromolar enzyme inhibition, high ligand efficiency, and also a novel scaffold.
Additionally, just about the most active compounds showed promising whole-cell antibacterial activity from various Gram-positive and Gram-negative species, such as the goal pathogen.
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