Structure activity relationship of N-1 substituted 1,5-naphthyrid-2-one analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-9).

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 µg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 µg/mL).

Daptomycin for Pediatric Gram-Positive Acute Hematogenous Osteomyelitis.

BACKGROUND: We prospectively evaluated efficacy and safety of daptomycin versus active comparator in children with acute hematogenous osteomyelitis (AHO). METHODS: Randomized, controlled, double-blind, global, multicenter, phase 3 trial. Patients 1-17 years of age with suspected/confirmed AHO requiring hospitalization and intravenous therapy were randomized 1:1 to intravenous daptomycin (once-daily, age-adjusted doses) or comparator (vancomycin, nafcillin or equivalent) ≥4 days, followed by oral therapy (14-42 days total). Primary endpoint: protocol-defined clinical improvement by Day 5 in the modified intention-to-treat (MITT) population (confirmed AHO, ≥1 dose of study treatment); differences between study arms were evaluated using a prespecified 15% noninferiority margin for daptomycin. RESULTS: Seventy-three patients per arm received treatment. Pathogens were isolated from 62% of patients (83% methicillin-susceptible Staphylococcus aureus, 9% methicillin-resistant S. aureus [MRSA]). Clinical improvement by Day 5 was observed in 55/71 (78%) daptomycin- and 58/70 (83%) comparator-treated MITT patients (95% confidence interval [CI]: -19.4, 7.4). This difference was not statistically significant; however, daptomycin did not meet the prespecified 15% noninferiority margin, since the lower bound of the 95% CI extended below 15%. Overall, 82% of daptomycin and 87% of comparator patients achieved clinical cure at the test-of-cure visit (secondary endpoint). More comparator patients had treatment-emergent (63% vs. 46%) and treatment-related (18% vs. 7%) adverse events. CONCLUSIONS: Differences between daptomycin and comparator for the primary endpoint were not statistically significant; however, prespecified noninferiority criteria for daptomycin were not met. With insufficient cases of confirmed MRSA, we could not evaluate daptomycin for MRSA AHO. Our nonvalidated protocol design yields valuable information for implementing future trials in AHO (ClinicalTrials.gov NCT01922011).

Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase.

To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.

Quantitation of wall teichoic acid in Staphylococcus aureus by direct measurement of monomeric units using LC-MS/MS.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for new therapeutic agents capable of combating this threat. We have previously reported on the discovery of novel inhibitors targeting enzymes involved in the biosynthesis of wall teichoic acid (WTA) and demonstrated that these agents can restore ß-lactam efficacy against MRSA. In those previous reports pathway engagement of inhibitors was demonstrated by reduction in WTA levels measured by polyacrylamide gel electrophoresis. To enable a more rigorous analysis of these inhibitors we sought to develop a quantitative method for measuring whole-cell reductions in WTA. Herein we describe a robust methodology for hydrolyzing polymeric WTA to the monomeric component ribitol-N-acetylglucosamine coupled with measurement by LC-MS/MS. Critical elements of the protocol were found to include the time and temperature of hydrofluoric acid-mediated hydrolysis of polymeric WTA and optimization of these parameters is fully described. Most significantly, the assay enabled accurate and reproducible measurement of depletion EC50s for tunicamycin and representatives from the novel class of TarO inhibitors, the tarocins. The method described can readily be adapted to quantifying levels of WTA in tissue homogenates from a murine model of infection, highlighting the applicability for both in vitro and in vivo characterizations.

Benzimidazole analogs as WTA biosynthesis inhibitors targeting methicillin resistant Staphylococcus aureus.

A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure-activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci.

In Vitro and In Vivo Characterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, and in vivo characterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli and displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergence in vitro at concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activity in vitro and in vivo.

NMR Binding and Functional Assays for Detecting Inhibitors of S. aureus MnaA.

Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated ß-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore ß-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrate and product (UDP-GlcNAc/UDP-ManNAc) share an identical molecular weight. Herein, we developed a nuclear magnetic resonance (NMR) functional assay that can be combined with other NMR approaches to triage putative MnaA inhibitors from phenotypic cell-based screening campaigns. In addition, we determined that tunicamycin, a potent WTA pathway inhibitor, inhibits both S. aureus MnaA and a functionally redundant epimerase, Cap5P.

Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6).

Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30 µM) profile.

Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-5).

Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property.

Hydroxy tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of RHS moiety (Part-3).

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. (R)-Hydroxy-1,5-naphthyridinone left-hand side (LHS) oxabicyclooctane linked pyridoxazinone right-hand side (RHS) containing NBTIs showed a potent Gram-positive antibacterial profile. SAR around the RHS moiety, including substitutions around pyridooxazinone, pyridodioxane, and phenyl propenoids has been described. A fluoro substituted pyridoxazinone showed an MIC against Staphylococcus aureus of 0.5 µg/mL with reduced functional hERG activity (IC50 333 µM) and good in vivo efficacy [ED90 12 mg/kg, intravenous (iv) and 15 mg/kg, oral (p.o.)]. A pyridodioxane-containing NBTI showed a S. aureus MIC of 0.5 µg/mL, significantly improved hERG IC50 764 µM and strong efficacy of 11 mg/kg (iv) and 5 mg/kg (p.o.). A phenyl propenoid series of compounds showed potent antibacterial activity, but also showed potent hERG binding activity. Many of the compounds in the hydroxy-tricyclic series showed strong activity against Acinetobacter baumannii, but reduced activity against Escherichia coli and Pseudomonas aeruginosa. Bicyclic heterocycles appeared to be the best RHS moiety for the hydroxy-tricyclic oxabicyclooctane linked NBTIs.

Tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of left-hand-side moiety (Part-2).

Novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. A series of novel oxabicyclooctane-linked NBTIs with new tricyclic-1,5-naphthyridinone left hand side moieties have been described. Compounds with a (R)-hydroxy-1,5-naphthyridinone moiety (7) showed potent antibacterial activity (e.g., Staphylococcus aureus MIC 0.25 µg/mL), acceptable Gram-positive and Gram-negative spectrum with rapidly bactericidal activity. The compound 7 showed intravenous and oral efficacy (ED50) at 3.2 and 27 mg/kg doses, respectively, in a murine model of bacteremia. Most importantly they showed significant attenuation of functional hERG activity (IC50 >170 µM). In general, lower logD attenuated hERG activity but also reduced Gram-negative activity. The co-crystal structure of a hydroxy-tricyclic NBTI bound to a DNA-gyrase complex exhibited a binding mode that show enantiomeric preference for R isomer and explains the activity and SAR. The discovery, synthesis, SAR and X-ray crystal structure of the left-hand-side tricyclic 1,5-naphthyridinone based oxabicyclooctane linked NBTIs are described.

Structure activity relationship of substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-4).

Bacterial resistance is rapidly growing, necessitating the need to discover new agents. Novel bacterial topoisomerase inhibitors (NBTIs) are new class of broad-spectrum antibacterial agents targeting bacterial DNA gyrase and topoisomerase IV. This class of inhibitors binds to an alternative binding site relative to fluoroquinolones and shows no cross-resistance to quinolones. NBTIs consist of three structural motifs. A structure activity relationship of the left hand motif 1,5-naphthyridine of oxabicyclooctane-linked NBTIs is described. Fifty five compounds were evaluated against a panel of key Gram-positive and Gram-negative strains of bacteria, as well as for hERG activity and five compounds were tested for in vivo efficacy in murine model of Staphylococcus aureus infection. These studies suggest that only a narrow range (activating and deactivating) of substitutions at C-2 and C-7 are tolerated for optimal antibacterial activity and spectrum. An alkoxy (methoxy) and CN at C-2, and a halogen and hydroxyl at C-7, appeared to be preferred in this series. Substitutions on the other three carbons generally have detrimental effect on the activity. No clear hERG activity SAR emerged from these substitutions.

Effect of suberoylanilide hydroxamic acid (SAHA) administration on the residual virus pool in a model of combination antiretroviral therapy-mediated suppression in SIVmac239-infected indian rhesus macaques.

Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4(+) T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches.

Drug/Cell-line Browser: interactive canvas visualization of cancer drug/cell-line viability assay datasets.

SUMMARY: Recently, several high profile studies collected cell viability data from panels of cancer cell lines treated with many drugs applied at different concentrations. Such drug sensitivity data for cancer cell lines provide suggestive treatments for different types and subtypes of cancer. Visualization of these datasets can reveal patterns that may not be obvious by examining the data without such efforts. Here we introduce Drug/Cell-line Browser (DCB), an online interactive HTML5 data visualization tool for interacting with three of the recently published datasets of cancer cell lines/drug-viability studies. DCB uses clustering and canvas visualization of the drugs and the cell lines, as well as a bar graph that summarizes drug effectiveness for the tissue of origin or the cancer subtypes for single or multiple drugs. DCB can help in understanding drug response patterns and prioritizing drug/cancer cell line interactions by tissue of origin or cancer subtype. AVAILABILITY AND IMPLEMENTATION: DCB is an open source Web-based tool that is freely available at: http://www.maayanlab.net/LINCS/DCB CONTACT: avi.maayan@mssm.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

LINCS Canvas Browser: interactive web app to query, browse and interrogate LINCS L1000 gene expression signatures.

For the Library of Integrated Network-based Cellular Signatures (LINCS) project many gene expression signatures using the L1000 technology have been produced. The L1000 technology is a cost-effective method to profile gene expression in large scale. LINCS Canvas Browser (LCB) is an interactive HTML5 web-based software application that facilitates querying, browsing and interrogating many of the currently available LINCS L1000 data. LCB implements two compacted layered canvases, one to visualize clustered L1000 expression data, and the other to display enrichment analysis results using 30 different gene set libraries. Clicking on an experimental condition highlights gene-sets enriched for the differentially expressed genes from the selected experiment. A search interface allows users to input gene lists and query them against over 100 000 conditions to find the top matching experiments. The tool integrates many resources for an unprecedented potential for new discoveries in systems biology and systems pharmacology. The LCB application is available at http://www.maayanlab.net/LINCS/LCB. Customized versions will be made part of the http://lincscloud.org and http://lincs.hms.harvard.edu websites.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad spectrum antibacterial agents.

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC50 > 18 µM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase (IC50 = 1.02 µM) and topo IV (IC50 = 10.4 µM). AM8191 showed parenteral and oral efficacy (ED50) at less than 2.5 mg/kg doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker.

Proteomics analysis of the non-muscle myosin heavy chain IIa-enriched actin-myosin complex reveals multiple functions within the podocyte.

MYH9 encodes non-muscle myosin heavy chain IIA (NMMHCIIA), the predominant force-generating ATPase in non-muscle cells. Several lines of evidence implicate a role for MYH9 in podocytopathies. However, NMMHCIIA's function in podocytes remains unknown. To better understand this function, we performed immuno-precipitation followed by mass-spectrometry proteomics to identify proteins interacting with the NMMHCIIA-enriched actin-myosin complexes. Computational analyses revealed that these proteins belong to functional networks including regulators of cytoskeletal organization, metabolism and networks regulated by the HIV-1 gene nef. We further characterized the subcellular localization of NMMHCIIA within podocytes in vivo, and found it to be present within the podocyte major foot processes. Finally, we tested the effect of loss of MYH9 expression in podocytes in vitro, and found that it was necessary for cytoskeletal organization. Our results provide the first survey of NMMHCIIA-enriched actin-myosin-interacting proteins within the podocyte, demonstrating the important role of NMMHCIIA in organizing the elaborate cytoskeleton structure of podocytes. Our characterization of NMMHCIIA's functions goes beyond the podocyte, providing important insights into its general molecular role.

Murgocil is a highly bioactive staphylococcal-specific inhibitor of the peptidoglycan glycosyltransferase enzyme MurG.

Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a ß-lactam potentiation screen to identify small molecules that augment the activity of ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the ß-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive ß-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.

ESCAPE: database for integrating high-content published data collected from human and mouse embryonic stem cells.

High content studies that profile mouse and human embryonic stem cells (m/hESCs) using various genome-wide technologies such as transcriptomics and proteomics are constantly being published. However, efforts to integrate such data to obtain a global view of the molecular circuitry in m/hESCs are lagging behind. Here, we present an m/hESC-centered database called Embryonic Stem Cell Atlas from Pluripotency Evidence integrating data from many recent diverse high-throughput studies including chromatin immunoprecipitation followed by deep sequencing, genome-wide inhibitory RNA screens, gene expression microarrays or RNA-seq after knockdown (KD) or overexpression of critical factors, immunoprecipitation followed by mass spectrometry proteomics and phosphoproteomics. The database provides web-based interactive search and visualization tools that can be used to build subnetworks and to identify known and novel regulatory interactions across various regulatory layers. The web-interface also includes tools to predict the effects of combinatorial KDs by additive effects controlled by sliders, or through simulation software implemented in MATLAB. Overall, the Embryonic Stem Cell Atlas from Pluripotency Evidence database is a comprehensive resource for the stem cell systems biology community. Database URL: http://www.maayanlab.net/ESCAPE

Network2Canvas: network visualization on a canvas with enrichment analysis.

MOTIVATION: Networks are vital to computational systems biology research, but visualizing them is a challenge. For networks larger than ∼100 nodes and ∼200 links, ball-and-stick diagrams fail to convey much information. To address this, we developed Network2Canvas (N2C), a web application that provides an alternative way to view networks. N2C visualizes networks by placing nodes on a square toroidal canvas. The network nodes are clustered on the canvas using simulated annealing to maximize local connections where a node's brightness is made proportional to its local fitness. The interactive canvas is implemented in HyperText Markup Language (HTML)5 with the JavaScript library Data-Driven Documents (D3). We applied N2C to visualize 30 canvases made from human and mouse gene-set libraries and 6 canvases made from the Food and Drug Administration (FDA)-approved drug-set libraries. Given lists of genes or drugs, enriched terms are highlighted on the canvases, and their degree of clustering is computed. Because N2C produces visual patterns of enriched terms on canvases, a trained eye can detect signatures instantly. In summary, N2C provides a new flexible method to visualize large networks and can be used to perform and visualize gene-set and drug-set enrichment analyses. AVAILABILITY: N2C is freely available at http://www.maayanlab.net/N2C and is open source. CONTACT: avi.maayan@mssm.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.