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ObjectiveSince previous studies indicate that metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), we undertook this study to determine if changes in the genome-wide chromatin and DNA states in genes associated with nutrient transporters could help to identify activated metabolic pathways in RA FLS.MethodsData from a previous comprehensive epigenomic study in FLS were analyzed to identify differences in genome-wide states and gene transcription between RA and osteoarthritis. We utilized the single nearest genes to regions of interest for pathway analyses. Homer promoter analysis was used to identify enriched motifs for transcription factors. The role of solute carrier transporters and glutamine metabolism dependence in RA FLS was determined by small interfacing RNA knockdown, functional assays, and incubation with CB-839, a glutaminase inhibitor. We performed 1 H nuclear magnetic resonance to quantify metabolites.ResultsThe unbiased pathway analysis demonstrated that solute carrier-mediated transmembrane transport was one pathway associated with differences in at least 4 genome-wide states or gene transcription. Thirty-four transporters of amino acids and other nutrients were associated with a change in at least 4 epigenetic marks. Functional assays revealed that solute carrier family 4 member 4 (SLC4A4) was critical for invasion, and glutamine was sufficient as an alternate source of energy to glucose. Experiments with CB-839 demonstrated decreased RA FLS invasion and proliferation. Finally, we found enrichment of motifs for c-Myc in several nutrient transporters.ConclusionOur findings demonstrate that changes in the epigenetic landscape of genes are related to nutrient transporters, and metabolic pathways can be used to identify RA-specific targets, including critical solute carrier transporters, enzymes, and transcription factors, to develop novel therapeutic agents.

Objective: \ud Fibroblast-like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase-8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs.\ud \ud Methods: \ud RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8. Caspase-8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase-8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase-8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase-8B and G were determined.\ud \ud Results: \ud Caspase-8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase-8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase-8) and induced by PDGF. The crystal structures of caspase-8 variant G and B were identical except for a unique unstructured 59 amino acid N-terminal domain in variant G. Selective knockdown of caspase-8G was solely responsible for the effects of caspase-8 on calpain activity and cell invasion in FLS.\ud \ud Conclusion: \ud Blocking caspase-8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase-8 inhibition.

Antibiotic and antimicrobial resistance is a continually growing challenge in the treatment of various bacterial infections worldwide. New drugs and new drug targets are necessary to curb the threat of infectious diseases caused by multidrug-resistant pathogens. The tryptophan biosynthesis pathway is essential for bacterial growth but is absent in higher animals and humans. Drugs that can inhibit the bacterial biosynthesis of tryptophan offer a new class of antibiotics. In this work, we combined a structure-based strategy using in silico docking screening and molecular dynamics (MD) simulations to identify compounds targeting the α subunit of tryptophan synthase with experimental methods involving the whole-cell minimum inhibitory concentration (MIC) test, solution state NMR, and crystallography to confirm the inhibition of L-tryptophan biosynthesis. Screening 1800 compounds from the National Cancer Institute Diversity Set I against α subunit revealed 28 compounds for experimental validation; four of the 28 hit compounds showed promising activity in MIC testing. We performed solution state NMR experiments to demonstrate that Compound 1 binds to the α subunit. We also report a crystal structure of Salmonella enterica serotype Typhimurium tryptophan synthase in complex with one successful inhibitor, 3-amino-3-imino-2-phenyldiazenylpropanamide (Compound 1) which revealed a binding site at the αβ interface of the dimeric enzyme. MD simulations were carried out to examine two binding sites for the compound. Our results show that this small molecule inhibitor could be a promising lead for future drug development. This article is protected by copyright. All rights reserved.

Objective Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) derived from hip and knee have distinctive DNA methylation and transcriptome patterns in interleukin (IL)-6 signaling and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. To determine the functional effects of these joint-specific signatures, we evaluated how RA hip and knee FLS differ in their response to IL-6. Methods Hip or knee RA FLS were obtained after arthroplasty. Previously published datasets on epigenetic landscape of FLS were mined to identify joint-specific IL-6-related epigenomic differences. RNA sequencing was performed on five RA hip and five knee FLS treated with or without IL-6. Differential gene expression was determined using edgeR software. STAT3 phosphorylation was measured using bead assays. Sensitivity to tofacitinib was evaluated by measuring CCL2 inhibition using quantitative polymerase chain reaction. Results Assay for Transposase-Accessible Chromatin sequencing and histone chromatin immunoprecipitation sequencing datasets from RA FLS were analyzed to identify epigenomic differences between hip and knee. Differential chromatin accessibility was associated with IL-6,IL-6R, and JAK1 genes. H3K27ac was also differentially marked at other JAK-STAT-related genes, including STAT3-STAT5A region. Principal component analysis of RNA sequencing data confirmed segregation between RA hip and knee FLS under basal conditions, that persisted following IL-6 treatment. STAT3 phosphorylation after IL-6 was significantly higher in knee than hip FLS and was highly correlated with JAK1 protein levels. Knee FLS were less sensitive to the JAK inhibitor tofacitinib than hip FLS. Conclusion RA hip and knee FLS have distinct transcriptomes, epigenetic marks, and STAT3 activation patterns in the IL-6 pathway. These joint-specific differences might contribute to a differential clinical response in individual joints to targeted therapies such as JAK inhibitors.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/β-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation., Fibroblast-like synoviocytes are important mediators of joint pathology in rheumatoid arthritis (RA). Here the authors show that Lasp1 is epigenetically regulated and highly expressed by these cells in RA and its deletion can limit joint pathology in a mouse model of inflammatory arthritis.

Immune checkpoint blockade (ICB) therapies are now established as first-line treatments for multiple cancers, but many patients do not derive long-term benefit from ICB. Here, we report that increased amounts of histone 3 lysine 4 demethylase KDM5A in tumors markedly improved response to the treatment with the programmed cell death protein 1 (PD-1) antibody in mouse cancer models. In a screen for molecules that increased KDM5A abundance, we identified one (D18) that increased the efficacy of various ICB agents in three murine cancer models when used as a combination therapy. D18 potentiated ICB efficacy through two orthogonal mechanisms: (i) increasing KDM5A abundance, which suppressed expression of the gene PTEN (encoding phosphatase and tensin homolog) and increased programmed cell death ligand 1 abundance through a pathway involving PI3K-AKT-S6K1, and (ii) activating Toll-like receptors 7 and 8 (TLR7/8) signaling pathways. Combination treatment increased T cell activation and expansion, CD103+ tumor-infiltrating dendritic cells, and tumor-associated M1 macrophages, ultimately enhancing the overall recruitment of activated CD8+ T cells to tumors. In patients with melanoma, a high KDM5A gene signature correlated with KDM5A expression and could potentially serve as a marker of response to anti-PD-1 immunotherapy. Furthermore, our results indicated that bifunctional agents that enhance both KDM5A and TLR activity warrant investigation as combination therapies with ICB agents.

PTPRS-targeted FLS-directed therapy synergizes with TNF inhibition in therapy of arthritis., Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase–mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

ObjectiveWe aimed to understand the role of the tyrosine phosphatase PTPN14—which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol—in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).MethodsGene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis.ResultsRA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFβ-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP—but not of a non-YAP-interacting PTPN14 mutant—enhanced SMAD reporter activity. YAP promoted TGFβ-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity.ConclusionIn RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.

DNA methylation is an important epigenetic mark but how its locus-specificity is decided in relation to DNA sequence is not fully understood. Here, we have analyzed 34 diverse whole-genome bisulfite sequencing datasets in human and identified 313 motifs, including 92 and 221 associated with methylation (methylation motifs, MMs) and unmethylation (unmethylation motifs, UMs), respectively. The functionality of these motifs is supported by multiple lines of evidence. First, the methylation levels at the MM and UM motifs are respectively higher and lower than the genomic background. Second, these motifs are enriched at the binding sites of methylation modifying enzymes including DNMT3A and TET1, indicating their possible roles of recruiting these enzymes. Third, these motifs significantly overlap with “somatic QTLs” (quantitative trait loci) of methylation and expression. Fourth, disruption of these motifs by mutation is associated with significantly altered methylation level of the CpGs in the neighbor regions. Furthermore, these motifs together with somatic mutations are predictive of cancer subtypes and patient survival. We revealed some of these motifs were also associated with histone modifications, suggesting a possible interplay between the two types of epigenetic modifications. We also found some motifs form feed forward loops to contribute to DNA methylation dynamics.

The Infinium HumanMethylation450 BeadChip array, referred as 450K array hereinafter, has been widely adopted as an affordable technique to determine DNA methylation. Tens of thousands of data have been generated on diverse cell types and patient tissues, which have provided great insight into understanding the crucial roles of epigenetic modifications in many biological processes and diseases. The limitation of this technique is its coverage, which measures methylation levels of about 450,000 CpGs, accounting for about 1.6% of all CpGs in the human genome. In the present study we developed and compared computational models to significantly expand the coverage of Illumina 450K (~11 folds). Using the whole genome bisulfite sequencing and Illumina 450K data in the human H1 embryonic stem cell, we showed that the predicted and measured methylation levels were well correlated. Our proposed model showed superior prediction accuracies compared to the existing methods on the same dataset. When applied to predict the DNA methylome on other cells, our proposed model achieved comparable performance in cross-validations, which indicates the generalizibility of the method. Our method would thus be invaluable to maximize the usage of the existing data.

Background:The “Targeting Immune Responses for Prevention of RA” (TIP-RA) collaboration studies individuals at high risk for developing RA because of serum anti-citrullinated protein antibody positivity in absence of arthritis, and is focused on defining how they transition from at-risk to classifiable disease. One potential mechanism is through alterations in epigenetics patterns in adaptive immune cells.Objectives:Previous studies showed that DNA methylation patterns of early RA (ERA) synoviocytes differ from long-standing RA, suggesting that abnormal methylation occurs early in synovium and evolves over time. To extend these observations, we performed a cross-sectional analysis in TIP-RA of DNA methylation signatures in peripheral blood cells in ERA, at-risk anti-CCP3+ individuals and demographically matched CCP- controls.Methods:Genomic DNA was isolated from two independent cohorts of CCP- (cohorts 1 and 2, respectively: B cell: n = 17/34; memory T cell: n = 21/34; and naïve T cell: n = 21/33), CCP3+ (B cell: n = 18/37; memory T cell: n = 20/36; and naïve T cell: n = 20/35), and CCP3+ ERA (B cell: n = 4/18; memory T cell: n = 5/18; and naïve T cell: n = 5/18) after separating PBMCs using antibodies and magnetic beads. Methylation was measured by Illumina Infinium MethylationEPIC chip. Differentially methylated loci (DMLs) were identified using Welch’s t-test and mapped to gene promoter regions to define DM genes (DMGs). Principal component analysis (PCA) was used to represent relationship among groups. Pathway analysis was applied by Reactome.Results:For the initial cohort, 1494, 1097 and 1330 DMLs were identified among CCP+, CCP- and ERA in B cells, memory T cells and naïve T cells, respectively. For the confirmatory cohort, 523, 793 and 548 DMLs were found in corresponding cell populations. The DML overlap between the 2 cohorts was highly significant (p= 2.48E-77). The DMLs were combined for both groups and corresponded to 411, 412, and 351 DMGs in B cells, memory T cells and naïve T cells. Of these, we found 246, 198 and 195 DMGs between CCP3+ and ERA in each peripheral blood cell population, respectively. PCA showed separation of CCP+, CCP- and ERA in each of the three blood cell types by DMLs (Fig. 1). DMGs were mapped to biological pathways to identify DM pathways. Although most were not significant, there were several highly significant differences comparing CCP+, ERA and CCP- in memory T cells involving pathways, including “Interferon gamma signaling” (FDR 7.48E-14), “PD-1 signaling” (FDR 8.71E-10), “Translocation of ZAP-70 to Immunological synapse” (FDR 4.75E-10), and “Phosphorylation of CD3 and TCR zeta chains” (FDR 8.71E-10).Figure 1.PCA shows the separation of CCP+, CCP- and ERA patients in memory T cells in confirmatory cohort.Conclusion:We identified reproducible methylation signatures of CCP-, CCP+, and ERA in peripheral blood B cells, memory T cells and naïve T cells in initial and confirmatory cohorts. The methylome of ERA also demonstrated a distinctive pattern from CCP+, indicating that progression to RA is accompanied by epigenetic remodeling, especially in T cell signaling and interferon responses. These signatures identify critical pathways in CCP positivity and classifiable RA and could provide the basis of novel interventions to prevent disease.Disclosure of Interests:Rizi Ai: None declared, David Boyle: None declared, Deepa Hammaker: None declared, Kevin Deane Grant/research support from: Janssen, Consultant of: Inova, ThermoFisher, Janseen, BMS and Microdrop, V. Michael Holers Grant/research support from: Janssen, Celgene, and BMS, Andre Matti: None declared, William Robinson: None declared, Jane Buckner Grant/research support from: Bristol-Myers Squibb, Janssen, Navin Rao Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Frederic Baribaud Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, Alyssa Johnsen Employee of: Janssen, Sunil Nagpal Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Wei Wang: None declared, Gary Firestein Grant/research support from: Lilly, Janssen, Abbvie

The integration of genomic and DNA methylation data has been demonstrated as a powerful strategy in understanding cancer mechanisms and identifying therapeutic targets. The TCGA consortium has mapped DNA methylation in thousands of cancer samples using Illumina Infinium Human Methylation 450 K BeadChip (Illumina 450 K array) that only covers about 1.5% of CpGs in the human genome. Therefore, increasing the coverage of the DNA methylome would significantly leverage the usage of the TCGA data. Here, we present a new model called EAGLING that can expand the Illumina 450 K array data 18 times to cover about 30% of the CpGs in the human genome. We applied it to analyze 13 cancers in TCGA. By integrating the expanded methylation, gene expression, and somatic mutation data, we identified the genes showing differential patterns in each of the 13 cancers. Many of the triple-evidenced genes identified in majority of the cancers are biomarkers or potential biomarkers. Pan-cancer analysis also revealed the pathways in which the triple-evidenced genes are enriched, which include well known ones as well as new ones, such as axonal guidance signaling pathway and pathways related to inflammatory processing or inflammation response. Triple-evidenced genes, particularly TNXB, RRM2, CELSR3, SLC16A3, FANCI, MMP9, MMP11, SIK1, and TRIM59 showed superior predictive power in both tumor diagnosis and prognosis. These results have demonstrated that the integrative analysis using the expanded methylation data is powerful in identifying critical genes/pathways that may serve as new therapeutic targets.

Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with “Huntington’s Disease Signaling” identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets., Fibroblast-like synoviocytes (FLS) in the intimal layer of the synovium can become invasive and destroy cartilage in patients with rheumatoid arthritis (RA). Here the authors integrate a variety of epigenomic data to map the epigenome of FLS in RA and identify potential therapeutic targets.

Background Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. Results Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5–10 molecules. Conclusions Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3300-3) contains supplementary material, which is available to authorized users.

Single-nucleus gene expression Identifying the genes expressed at the level of a single cell nucleus can better help us understand the human brain. Blue et al. developed a single-nuclei sequencing technique, which they applied to cells in classically defined Brodmann areas from a postmortem brain. Clustering of gene expression showed concordance with the area of origin and defining 16 neuronal subtypes. Both excitatory and inhibitory neuronal subtypes show regional variations that define distinct cortical areas and exhibit how gene expression clusters may distinguish between distinct cortical areas. This method opens the door to widespread sampling of the genes expressed in a diseased brain and other tissues of interest. Science , this issue p. 1586

Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate the measurement transfer functions to be linear above ~5-10 molecules. Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

Motivation: DNA methylation signatures in rheumatoid arthritis (RA) have been identified in fibroblast-like synoviocytes (FLS) with Illumina HumanMethylation450 array. Since Results: We developed a computational model that is trained on 14 tissues with both whole genome bisulfite sequencing and 450K array data. This model integrates information derived from the similarity of local methylation pattern between tissues, the methylation information of flanking CpG sites and the methylation tendency of flanking DNA sequences. The predicted and measured methylation values were highly correlated with a Pearson correlation coefficient of 0.9 in leave-one-tissue-out cross-validations. Importantly, the majority (76%) of the top 10% differentially methylated loci among the 14 tissues was correctly detected using the predicted methylation values. Applying this model to 450K data of RA, osteoarthritis and normal FLS, we successfully expanded the coverage of CpG sites 18.5-fold and accounts for about 30% of all the CpGs in the human genome. By integrative omics study, we identified genes and pathways tightly related to RA pathogenesis, among which 12 genes were supported by triple evidences, including 6 genes already known to perform specific roles in RA and 6 genes as new potential therapeutic targets. Availability and implementation: The source code, required data for prediction, and demo data for test are freely available at: http://wanglab.ucsd.edu/star/LR450K/. Contact: wei-wang@ucsd.edu or gfirestein@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Gene detection outliers. Genes that are problematic for detection. See Methods for classification of outliers. â Gene setâ indicates whether gene is classified as computationally unambiguous (1) or not (2). â Detected/undetectedâ indicates whether the gene is unexpectedly observed (D) or unexpectedly unobserved (U). (PDF 181 kb)

Computationally unambiguous genes. Genes to which reads can be uniquely assigned. See the Excluded and unambiguous genes section in Methods for details on classification. (PDF 516 kb)

Accuracy and robustness of estimated reference HBR and UHR RNA expression levels. A. Consistency of abundance estimates by three quantification algorithms relative to publicly available PrimePCR measurements (see Methods ). Scatters show log10 reads per million (HTSeq [22] and Maxcounts [31]), log10 transcripts per million (RSEM), or log10 molecules (PrimePCR). Upper quadrants indicate Pearson correlation (R) of log-transformed estimates. Pairwise zeros were treated as missing values. Estimates were based on combined raw reads from 3 bulk reference samples generated using ribosomal depletion for each HBR and UHR. RSEM estimates were used as reference throughout. B. Accuracy and robustness of expression estimates across library preparation methods: ribosomal-depletion (combined n = 3 samples per HBR and UHR) and poly-A RNA selection (combined n = 4 samples per source). See Methods for sample information. Scatters as in A using RSEM expression level estimates for each library preparation method. Ribosomal-depletion samples were used as reference throughout. Abbreviations: Human Brain Reference (HBR), Universal Human Reference RNA (UHR). (PDF 469 kb)

Cannabinoid (CB1) receptor is a therapeutic drug target, and its structure and conformational changes after ligand binding are of great interest. To study the protein conformations in ligand bound state and assist in drug discovery, CB1 receptor homology models are needed for computer-based ligand screening. The known CB1 ligands are highly diverse structurally, so CB1 receptor may undergo considerable conformational changes to accept different ligands, which is challenging for molecular docking methods. To account for the flexibility of CB1 receptor, we constructed four CB1 receptor models based on four structurally distinct ligands, HU-210, ACEA, WIN55212-2 and SR141716A, using the newest X-ray crystal structures of human β₂ adrenergic receptor and adenosine A(2A) receptor as templates. The conformations of these four CB1-ligand complexes were optimized by molecular dynamics (MD) simulations. The models revealed interactions between CB1 receptor and known binders suggested by experiments and could successfully discriminate known ligands and non-binders in our docking assays. MD simulations were used to study the most flexible ligand, ACEA, in its free and bound states to investigate structural mobility achieved by the rearrangement of the fatty acid chain. Our models may capture important conformational changes of CB1 receptor to help improve accuracy in future CB1 drug screening.

Cannabinoids represent a promising class of compounds for developing novel therapeutic agents. Since the isolation and identification of the major psychoactive component Δ(9)-THC in Cannabis sativa in the 1960s, numerous analogues of the classical plant cannabinoids have been synthesized and tested for their biological activity. These compounds primarily target the cannabinoid receptors 1 (CB1) and Cannabinoid receptors 2 (CB2). This chapter focuses on CB1. Despite the lack of crystal structures for CB1, protein-based homology modeling approaches and molecular docking methods can be used in the design and discovery of cannabinoid analogues. Efficient synthetic approaches for therapeutically interesting cannabinoid analogues have been developed to further facilitate the drug discovery process.

Cannabinoids represent a promising class of compounds for developing novel therapeutic agents. Since the isolation and identification of the major psychoactive component D 9 -THC in Cannabis sativa in the 1960s, numerous analogues of the classical plant cannabinoids have been synthesized and tested for their biological activity. These compounds primarily target the cannabinoid receptors 1 (CB1) and Cannabinoid receptors 2 (CB2). This chapter focuses on CB1. Despite the lack of crystal structures for CB1, protein-based homology modeling approaches and molecular docking methods can be used in the design and discovery of cannabinoid analogues. Efficient synthetic approaches for therapeutically interesting cannabinoid analogues have been developed to further facilitate the drug discovery process.

Purpose The thermodynamically favored complex between the nuclear vitamin D receptor (VDR) and 1α,25(OH)2-vitamin D3 (1,25D3) triggers a shift in equilibrium to favor VDR binding to DNA, heterodimerization with the nuclear retinoid x receptor (RXR) and subsequent regulation of gene transcription. The key amino acids and structural requirements governing VDR binding to nuclear coactivators (NCoA) are well defined. Yet very little is understood about the internal changes in amino acid flexibility underpinning the control of ligand affinity, helix 12 conformation and function. Herein, we use molecular dynamics (MD) to study how the backbone and side-chain flexibility of the VDR differs when a) complexed to 1α,25(OH)2-vitamin D3 (1,25D3, agonist) and (23S),25-dehydro-1α(OH)-vitamin D3-26,23-lactone (MK, antagonist); b) residues that form hydrogen bonds with the C25-OH (H305 and H397) of 1,25D3 are mutated to phenylalanine; c) helix 12 conformation is changed and ligand is removed; and d) x-ray water near the C1- and C3-OH groups of 1,25D3 are present or replaced with explicit solvent. Methods We performed molecular dynamic simulations on the apo- and holo-VDRs and used T-Analyst to monitor the changes in the backbone and side-chain flexibility of residues that form regions of the VDR ligand binding pocket (LBP), NCoA surface and control helix 12 conformation. Results The VDR-1,25D3 and VDR-MK MD simulations demonstrate that 1,25D3 and MK induce highly similar changes in backbone and side-chain flexibility in residues that form the LBP. MK however did increase the backbone and side-chain flexibility of L404 and R274 respectively. MK also induced expansion of the VDR charge clamp (i.e. NCoA surface) and weakened the intramolecular interaction between H305---V418 (helix 12) and TYR401 (helix 11). In VDR_FF, MK induced a generally more rigid LBP and stronger interaction between F397 and F422 than 1,25D3, and reduced the flexibility of the R274 side-chain. Lastly the VDR MD simulations indicate that R274 can sample multiple conformations in the presence of ligand. When the R274 is extended, the β-OH group of 1,25D3 lies proximal to the backbone carbonyl oxygen of R274 and the side-chain forms H-bonds with hinge domain residues. This differs from the x-ray, kinked geometry, where the side-chain forms an H-bond with the 1α-OH group. Furthermore, 1,25D3, but not MK was observed to stabilize the x-ray geometry of R274 during the > 30 ns MD runs. Conclusions The MD methodology applied herein provides an in silico foundation to be expanded upon to better understand the intrinsic flexibility of the VDR and better understand key side-chain and backbone movements involved in the bimolecular interaction between the VDR and its’ ligands.