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Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.

Prostate-specific antigen (PSA) screening for prostate cancer (PCa) is limited by the lack of specificity but is further complicated in the benign prostatic hyperplasia (BPH) population which also exhibit elevated PSA, representing a clear unmet need to distinguish BPH from PCa. Herein, we evaluated the utility of FLNA IP-MRM, age, and prostate volume to stratify men with BPH from those with PCa. Diagnostic performance of the biomarker panel was better than PSA alone in discriminating patients with negative biopsy from those with PCa, as well as those who have had multiple prior biopsies (AUC 0.75 and 0.87 compared to AUC of PSA alone 0.55 and 0.57 for patients who have had single compared to multiple negative biopsies, respectively). Of interest, in patients with PCa, the panel demonstrated improved performance than PSA alone in those with Gleason scores of 5–7 (AUC 0.76 vs. 0.56) and Gleason scores of 8–10 (AUC 0.74 vs. 0.47). With Gleason scores (8–10), the negative predictive value of the panel is 0.97, indicating potential to limit false negatives in aggressive cancers. Together, these data demonstrate the ability of the biomarker panel to perform better than PSA alone in men with BPH, thus preventing unnecessary biopsies.

Corticotrophinomas represent 10% of all surgically removed pituitary adenomas, however, current treatment options are often not effective, and there is a need for improved pharmacological treatments. Recently, JQ1+, a bromodomain inhibitor that promotes gene transcription by binding acetylated histone residues and recruiting transcriptional machinery, has been shown to reduce proliferation in a murine corticotroph cell line, AtT20. RNA-Seq analysis of AtT20 cells following treatment with JQ1+ identified the calcium-sensing receptor (CaSR) gene as significantly downregulated, which was subsequently confirmed using real-time PCR and Western blot analysis. CaSR is a G protein-coupled receptor that plays a central role in calcium homeostasis but can elicit non-calcitropic effects in multiple tissues, including the anterior pituitary where it helps regulate hormone secretion. However, in AtT20 cells, CaSR activates a tumour-specific cAMP pathway that promotes ACTH and PTHrP hypersecretion. We hypothesised that the Casr promoter may harbour binding sites for BET proteins, and using chromatin immunoprecipitation (ChIP)-sequencing demonstrated that the BET protein Brd3 binds to the promoter of the Casr gene. Assessment of CaSR signalling showed that JQ1+ significantly reduced Ca2+e-mediated increases in intracellular calcium (Ca2+i) mobilisation and cAMP signalling. However, the CaSR-negative allosteric modulator, NPS-2143, was unable to reduce AtT20 cell proliferation, indicating that reducing CaSR expression rather than activity is likely required to reduce pituitary cell proliferation. Thus, these studies demonstrate that reducing CaSR expression may be a viable option in the treatment of pituitary tumours. Moreover, current strategies to reduce CaSR activity, rather than protein expression for cancer treatments, may be ineffective.

Rationale Hypertension, obesity and diabetes are major risk factors associated with morbidities underlying COVID-19 infections. Regression analysis correlated presence of ACE insertion/deletion (I/D) polymorphism to COVID-19 incidence and mortality. Furthermore, COVID-19 prevalence correlated to allele frequency of angiotensin-converting enzyme (ACE) deletion (D) polymorphism within the European population. Objective Homozygous ACE deletion polymorphism is associated with increase in ACE and angiotensin II (Ang-II), sustained levels can result in inflammation, fibrosis and organ damage. The ACE DD polymorphism is also associated with hypertension, acute respiratory distress and diabetic nephropathy, all considered high risk for COVID-19 infection and outcomes. The study objective was to describe a biological framework associating ethnic prevalence of ACE deletion polymorphism to COVID-19 comorbidities providing rationale for therapeutic utility of ACE-I/ARBs to improve outcomes. Method and Results The Allele Frequency Database (ALFRED) was queried for frequency of rs4646994 representing ACE I/D polymorphism. In a total of 349 worldwide population samples, frequency of ACE D allele was higher in European, Asian, and Africans cohorts. In the USA, the frequency of ACE D allele was higher in non-Hispanic Black compared with non-Hispanic White and Mexican Americans. Conclusion COVID-19 binding mediated reduction/inactivation of ACE-II can increase ACE/Ang-II signalling pathway and related pathologies. The presence of ACE DD polymorphism with COVID-19 infection likely augments ACE/Ang-II activities, increasing severity of COVID-19 morbidities and impacts outcomes. Thus, ethnic prevalence of ACE DD polymorphism can explain in part the severity of COVID-19 morbidity providing rationale for the use of ACE-I/ARBs to improve outcomes.

Neuroendocrine neoplasms (NENs) occur usually as sporadic tumours; however, rarely, they may arise in the context of a hereditary syndrome, such as multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder characterised by the combined development of pancreatic NENs (pNENs) together with parathyroid and anterior pituitary tumours. The therapeutic decision for sporadic pNENs patients is multi-disciplinary and complex: based on the grade and stage of the tumor, various options (and their combinations) are considered, such as surgical excision (either curative or for debulking aims), biological drugs (somatostatin analogues), targeted therapies (mTOR inhibitors or tyrosine kinases (TK)/receptors inhibitors), peptide receptor radioligand therapy (PRRT), chemotherapy, and liver-directed therapies. However, treatment of MEN1-related NENs’ patients is even more challenging, as these tumours are usually multifocal with co-existing foci of heterogeneous biology and malignant potential, rendering them more resistant to the conventional therapies used in their sporadic counterparts, and therefore associated with a poorer prognosis. Moreover, clinical data using standard therapeutic options in MEN1-related NENs are scarce. Recent preclinical studies have identified potentially new targeted therapeutic options for treating MEN1-associated NENs, such as epigenetic modulators, Wnt pathway-targeting β-catenin antagonists, Ras signalling modulators, Akt/mTOR signalling modulators, novel somatostatin receptors analogues, anti-angiogenic drugs, as well as MEN1 gene replacement therapy. The present review aims to summarize these novel therapeutic opportunities for NENs developing in the context of MEN1 syndrome, with an emphasis on pancreatic NENs, as they are the most frequent ones studied in MEN1-NENs models to date; moreover, due to the recent shifting nomenclature of ‘pituitary adenomas’ to ‘pituitary neuroendocrine neoplasms’, relevant data on MEN1-pituitary tumours, when appropriate, are briefly described.

Corticotrophinomas, which are neuroendocrine tumors (NETs) and represent >10% of all surgically removed pituitary adenomas, cause Cushing’s disease that is associated with hypersecretion of adrenocorticotropic hormone (ACTH) leading to excessive production of glucocorticoids by the adrenal cortex. Trans-sphenoidal hypophysectomy is the treatment of choice for corticotrophinomas, with medical treatments being reserved for patients who have contraindications for surgery. However, these treatments are often ineffective. Improved therapeutic approaches are required, and we have therefore assessed the efficacy of epigenetic modulators, a new class of anti-cancer drugs that have been reported to be effective in treating pancreatic NETs. Specifically, we assessed the anti-proliferative and pro-apoptotic effects of the bromo and extra terminal domain (BET) inhibitors JQ1 and PFI-1, in the mouse corticotrophinoma cell line AtT20, using Cell Titer Blue and Caspase Glo assays, respectively. JQ1, after 96h treatment, was more efficacious than PFI-1. Thus, JQ1 significantly decreased proliferation by 95%, (p50-fold (p

Introduction: Classification of breast cancer can incorporate immunohistochemical (IHC) detection of ER/PR/HER2/KI67 to stratify the subtypes. High throughput proteomics analysis allows for the expansion of biomarker discovery within the subtypes. We evaluated a cohort of 109 tumors characterized as ER+ (Luminal A and Luminal B1; HER2+ and ER low (1-10%) cases were excluded) compared to ER-/HER2- tumors. Utilizing an integrated bioinformatics approach, we developed a proteomic marker signature to reclassify tumors into ER+(like) and ER-(like) tumors. CPTAC (Proteomic)/TCGA (RNAseq) datasets and larger METBRIC and GSE96058 cohorts were used to validate this marker signature. The selected biomarkers demonstrated significant differences impacting survival outcome. Methods: Clinical IHC subtyping of core biopsies was used to select a cohort of patients with ER+/HER2- and ER-/HER2- primary tumors from flash-frozen surgical samples. The positive/negative status of ER/PR/HER2 was defined using updated ASCO 2020 guidelines. Ki-67 status was determined using the 2011 St. Gallen's International Expert Consensus recommendations. Proteomic analysis was performed using Thermo Q-Exactive+ LC MS/MS analysis. Differential analysis was applied to select the significantly altered proteins between ER+ and ER- cases, Univariate survival analysis was engaged to filter informative protein/genes using TCGA RNA-Seq data. Nearest centroid analysis was deployed to define the classifier to predict novel molecular subtypes. Results/Conclusions: We selected 34 proteins/genes from 164 significantly differentially expressed proteins for further analysis. The centroid model constructed with the 34 proteins defined 2 groups: ER+(like) and ER-(like). An additional 4 groups were defined across subtypes: luminal tumors classified both by IHC and marker signature (LL), luminal tumors classified by IHC but marker signature more like triple negative (LT), triple negative tumors classified by IHC but marker signature more like luminal (TL), and triple negative classified by both IHC and marker signature (TT). This marker signature segregated close to 5000 tumors across CPTAC, TCGA, METABRIC and GSE96058 cohorts. Survival analysis in these groups of patients revealed differences in radiation, hormone/radiation, hormone therapy, and hormone/radiation/chemotherapy treatments. In summary using proteomics data we identified a 34 gene/protein marker signature, validated in large external cohorts and exhibited impact on survival and response to therapy. Further, this signature was enriched in metabolism and microenvironmental associated factors that could represent novel targets or development combination strategies based on this signature. Citation Format: Guisong Wang, Punit Shah, Rick Searfoss, Leigh Fantacone-Campbell, Jeffrey A. Hooke, Brenda Deyarmin, Rebecca N. Zingmark, Stella Somiari, Jianfang Liu, Leonid Kvecher, Bradley Mostoller, Lori A. Sturtz, Praven-Kumar Raj-Kumar, Elder Granger, Linda Vahdat, Mary L. Cutler, Chas Bountra, Rangaprasad Sarangarajan, Hai Hu, Michael A. Kiebish, Albert J. Kovatich, Niven R. Narain, Craig D. Shriver. Reclassification of ER+ (luminal A/luminal B1 minus ER low)-like and ER- like breast tumors based on proteomic/gene and clinical outcome signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1188.

Introduction: In the United States, breast cancer represents the leading cancer diagnosis among women and can readily be classified as a metabolic disease based on its distinct metabolic activity within the tumor microenvironment. Compared to other omics technologies, extensive lipidomic and metabolomic studies are lacking. Here in, we evaluated a cohort of 109 tumors characterized as ER+/HER2- and ER-/HER2- based on immunohistochemistry (IHC) and performed comprehensive structural lipidomic, signaling lipidomic, and global metabolomic analyses for an extensive characterization of the biophysical, signaling, and metabolic interplay between these tumors. Methods: Clinical IHC subtyping of core biopsies was used to select a cohort of patients with ER+/HER2- or ER-/HER2- primary tumors from flash-frozen surgical samples. The positive/negative status of ER/PR/HER2 was defined using updated ASCO 2020 guidelines. Ki-67 status was determined using the 2011 St. Gallen's International Expert Consensus recommendations. ER low (1-10%) cases were excluded from this analysis. Structural lipidomic analysis was employed through the use of MS/MSALL high resolution shotgun lipidomics using a SCIEX 5600+ TripleTOF micro LC approach characterizing 23 lipid classes and over 1200 molecular species. Signaling lipids were analyzed using a SCIEX 6600 TripleTOF microLC platform characterizing 106 lipid analytes across octadecanoid, eicosanoid and docosanoid species. Metabolomics analysis was performed using LECO PEGASUS GC TOF, SCIEX 5500 HILIC LC MS/MS analysis, and SCIEX 6600 High resolution RP-LC-MS analysis detecting 450 metabolite Metabolomics data was further interpreted using MetaboAnalyst software. Results/Conclusions: Compared to their ER+ counterparts, ER-/HER2- tumors exhibited a significant decrease in triacylglycerides, and a corresponding increase in cholesterol ester, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol species demonstrating a signature of biophysical and metabolic rewiring with alterations in Kennedy pathway lipid shuttling. One signaling lipid was decreased and six were increased (predominantly arachidonic species) in ER-/HER2- tumors compared to ER+/HER2- ones. Metabolomic analysis revealed distinct alterations in cysteine/methionine, arginine/proline, purine, butanoate, and tryptophan metabolism. Utilizing a multidimensional metabolic integration approach, we identified distinct biophysical, signaling, and biochemical alterations in ER+/HER2- compared to ER-/HER2- breast tumors, which may impact selection of therapy and outcome in the future. Citation Format: Valerie Bussberg, Vladimir Tolstikov, Guisong Wang, Punit Shah, Rick Searfoss, Leigh Fantacone-Campbell, Jeffrey A. Hooke, Brenda Deyarmin, Rebecca N. Zingmark, Stella Somiari, Jianfang Liu, Leonid Kvecher, Bradley Mostoller, Lori Sturtz, Praveen-Kumar Raj-Kumar, Elder Granger, Linda Vahdat, Mary L. Cutler, Chas Bountra, Rangaprasad Sarangarajan, Hai Hu, Albert J. Kovatich, Michael A. Kiebish, Niven R. Narain, Craig D. Shriver. Multidimensional metabolomic stratification of ER+/HER2- compared to ER-/HER2- breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2342.

Background The emergence of COVID-19 progressed into a global pandemic that has functionally put the world at a standstill and catapulted major healthcare systems into an overburdened state. The dire need for therapeutic strategies to mitigate and successfully treat COVID-19 is now a public health crisis with national security implications for many countries.Methods The current study employed Bayesian networks to a longitudinal proteomic dataset generated from Caco-2 cells transfected with SARS-CoV-2 (isolated from patients returning from Wuhan to Frankfurt) [1]. Two different approaches were employed to assess the Bayesian models, a titer-center topology analysis and a drug signature enrichment analysis.Results Topology analysis identified a set of proteins directly linked to the SAR-CoV2 titer, including ACE2, a SARS-CoV-2 binding receptor, MAOB and CHECK1. Aligning with the topology analysis, MAOB and CHECK1 were also identified within the enriched drug-signatures.Conclusions Taken together, the data output from this network has identified nodal host proteins that may be connected to 18 chemical compounds, some already marketed, which provides an immediate opportunity to rapidly triage these assets for safety and efficacy against COVID-19.

The neoplastic transformation of cells and inflammation are processes that contribute to tumor initiation. Recently, emerging evidence has suggested that epigenetic alterations are also implicated in the early stages of carcinogenesis. Therefore, potent small molecules targeting epigenetic regulators have been developed as novel cancer therapeutic and preventive strategies. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that play key roles at the interface between chromatin modification and transcriptional regulation. In this study, we investigated the effect of the BET inhibitor JQ-1 on malignant transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 P+ cells. Treatment with JQ-1 effectively impaired TPA-induced colony formation in vitro. At the molecular level, the expression of several key TPA-induced pro-survival and pro-proliferative genes (Bcl2, Cyclin D1, and c-Myc) decreased rapidly after BET inhibition. In addition, JQ-1 treatment attenuated the activation of inflammatory NF-κB signaling triggered by TPA. Luciferase reporter assays using plasmids carrying different elements from the COX2 or IL6 promoters demonstrated that JQ-1 does not directly inhibit interactions between NF-κB and its binding sequence; rather, it affects CRE-element-associated transcriptional enhancement. Through siRNA gene silencing, we found that JQ-1 inhibits the p300-dependent transcriptional activation of COX2, which correlates with the results of the luciferase assay. Chromatin immunoprecipitation assays showed that TPA elevated H3K27Ac enrichment in the COX2 promoter region, which is mediated by p300, and Brd4. JQ-1 treatment did not change H3K27Ac levels but decreased the recruitment of Brd4 and RNA Polymerase II. Collectively, our study reveals that the BET inhibitor JQ-1 exerts potent anti-cancer and anti-inflammatory effects by interfering with the core transcriptional program of neoplastic transformation.

Precision medicine utilizes tailored diagnostic, prognostic and therapeutic strategies based on an individual’s molecular profile. Although it is gaining considerable traction and high-level political endorsement, it must overcome a number of translational hurdles, including regulatory barriers. At the core of precision medicine lies diagnostic tests and devices, however the regulatory classification of such products varies on a global basis. Navigating these convoluted regulatory pathways can be challenging - exacerbated by asymmetric technological advancement and regulatory progression. Both the EU and US are attempting to address such issues and newer concerns relating to direct-to-consumer testing. Flexible solutions are required to establish regulatory compliance across multiple countries and coordinated cross-collaboration initiatives need to empower technological development and globally harmonized regulation. The wider infrastructure, spanning beyond regulation, must also accommodate these changes and support subsequent clinical adoption, in order to firmly establish precision medicine in modern day medical practice.

Biomarker analysis is critically dependent on the quality of biofluid or tissue samples obtained from human research studies. Although proteomic, lipidomic, and metabolomic analyses can be dramatically impacted by the time of sample collection, fasting status, and participant demographics, the hemolysis status of plasma, serum or buffy coat samples is a poorly understood confounder of sample quality. Hemoglobin levels can range between 0 - 10 g/L in samples (referred to as 1-4 in plasma/serum and 0-4 level in buffy coat) as a marker of hemolysis severity and sample contamination by reticulocyte-derived analytes. In oncology clinical trials, patients can be more susceptible to hemolysis due to chemotherapy treatment, which can impact sample assessment and study results. Herein, we analyzed 941 plasma and 950 serum samples using comprehensive proteomics, structural lipidomics, signaling lipidomics, and metabolomics in a pancreatic cancer biomarker discovery program referred to as Project Survival as well as 951 buffy coat samples using only proteomic analysis. Project survival is a 7-year longitudinal pancreatic cancer biomarker discovery trial analyzing 400+ pancreatic cancer and at-risk patients using multi-omic and multiple biofluid assessment. To date this study yielded samples in plasma with 92.3% - #1, 6.5% - #2, 1.2% - #3, and 0% - #4 hemolysis, serum with 94.8% - #1, 3.8% - #2, 1.4% - #3, and 0% - #4 hemolysis and buffy coat 42.7% - #0, 25.6% - #1, 20.8% -#2, 10.4% - #3, and 0.5% - #4 hemolysis. Multi-omic and regression analysis of sample data for hemolysis status revealed a distinct pattern of OMIC variables correlated with the degree of hemolysis. Proteomics analysis was the greatest impacted in terms of the protein identification and quantitation. Additionally, pathway analysis revealed expected pathways associated with hemolysis and coagulation, but also unknown pathways and corresponding proteins that were differentially correlated with hemolysis state. Additionally, metabolomics and lipidomics analysis also revealed distinct differentials associated with hemolysis state. Herein, our analysis is the first to analyze thousands of samples using multi-omics revealing critically informative molecular differentials across OMIC technologies demonstrating that caution should be given to avoid these identified biomarkers for translational development. Citation Format: Michael A. Kiebish, Punit Shah, Valerie Bussberg, Vladimir Tolstikov, Rick Searfoss, Kennedy Ofori-Mensa, Eric M. Grund, Abena Darkwah, Emily Y. Chen, Bennett Greenwood, Ellaine Adu Ntoso, Leonardo Rodrigues, Mia Liu, Elder Granger, Chas Bountra, Rangaprasad Sarangarajan, A J. Moser, Niven R. Narain. Impact of hemolysis on multi-omic pancreatic cancer biomarker discovery: De-risking precision medicine biomarker development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2860.

The pressures on the pharmaceutical industry have incentivised a number of new collaborative models of research and development which can be categorised as open innovation. Examples of the different types of models employed are discussed and some, but not all, of these have been used to promote research and drug discovery for central nervous system disorders. Some are completely open access, while others have some intellectual property restrictions. Going forward, more ways of promoting open innovation and the sharing of best practice, especially in the neurosciences, will stimulate research and hopefully accelerate new medicines development.

Background\ud \ud Enhancer of zeste 2 (EZH2) promotes prostate cancer progression. We hypothesized that increased EZH2 expression is associated with postradiotherapy metastatic disease recurrence, and may promote radioresistance.\ud \ud \ud \ud Methods\ud \ud EZH2 expression was investigated using immunohistochemistry in diagnostic prostate biopsies of 113 prostate cancer patients treated with radiotherapy with curative intent. Associations between EZH2 expression in malignant and benign tissue in prostate biopsy cores and outcomes were investigated using univariate and multivariate Cox regression analyses. LNCaP and PC3 cell radiosensitivity was investigated using colony formation and γH2AX assays following UNC1999 chemical probe‐mediated EZH2 inhibition.\ud \ud \ud \ud Results\ud \ud While there was no significant association between EZH2 expression and biochemical recurrence following radiotherapy, univariate analysis revealed that prostate cancer cytoplasmic and total EZH2 expression were significantly associated with metastasis development postradiotherapy (P = 0.034 and P = 0.003, respectively). On multivariate analysis, the prostate cancer total EZH2 expression score remained statistically significant (P = 0.003), while cytoplasmic EZH2 expression did not reach statistical significance (P = 0.053). No association was observed between normal adjacent prostate EZH2 expression and biochemical recurrence or metastasis. LNCaP and PC3 cell treatment with UNC1999 reduced histone H3 lysine 27 tri‐methylation levels. Irradiation of LNCaP or PC3 cells with a single 2 Gy fraction with UNC1999‐mediated EZH2 inhibition resulted in a statistically significant, though modest, reduction in cell colony number for both cell lines. Increased γH2AX foci were observed 24 hours after ionizing irradiation in LNCaP cells, but not in PC3, following UNC1999‐mediated EZH2 inhibition vs controls.\ud \ud \ud \ud Conclusions\ud \ud Taken together, these results reveal that high pretreatment EZH2 expression in prostate cancer in diagnostic biopsies is associated with an increased risk of postradiotherapy metastatic disease recurrence, but EZH2 function may only at most play a modest role in promoting prostate cancer cell radioresistance.

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein–protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9+ acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers. Cancer Res; 75(23); 5106–19. ©2015 AACR.

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (ext-link ext-link-type="uri" xlink:href="https://openscienceprobes.sgc-frankfurt.de"https://openscienceprobes.sgc-frankfurt.de/ext-linkext-link ext-link-type="uri" xlink:href="https://openscienceprobes.sgc-frankfurt.de/"//ext-link). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.

Combination therapy has proven to be an effective strategy for treating many of the world's most intractable diseases. A growing number of investigators in academia, industry, regulatory agencies, foundations and advocacy organizations are interested in pursuing a combination approach to treating Alzheimer's disease. A meeting co-hosted by the Accelerate Cure/Treatments for Alzheimer's Disease Coalition, the Critical Path Institute and the Alzheimer's Association addressed challenges in designing clinical trials to test multiple treatments in combination and outlined a roadmap for making such trials a reality.

Background Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. Results A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. Conclusions High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0116-6) contains supplementary material, which is available to authorized users.

Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide - binding site and to the bottom of the chamber.

Discovering new medicines is difficult and increasingly expensive. The pharmaceutical industry has responded to this challenge by embracing open innovation to access external ideas. Historically, partnerships were usually bilateral, and the drug discovery process was shrouded in secrecy. This model is rapidly changing. With the advent of the Internet, drug discovery has become more decentralised, bottom-up, and scalable than ever before. The term open innovation is now accepted as just one of many terms that capture different but overlapping levels of openness in the drug discovery process. Many pharmaceutical companies recognise the advantages of revealing some proprietary information in the form of results, chemical tools, or unsolved problems in return for valuable insights and ideas. For example, such selective revealing can take the form of openly shared chemical tools to explore new biological mechanisms or by publicly admitting what is not known in the form of an open call. The essential ingredient for addressing these problems is access to the wider scientific crowd. The business of crowdsourcing, a form of outsourcing in which individuals or organisations solicit contributions from Internet users to obtain ideas or desired services, has grown significantly to fill this need and takes many forms today. Here, we posit that open-innovation approaches are more successful when they establish a reliable framework for converting creative ideas of the scientific crowd into practice with actionable plans.

We are experiencing a new era enabled by unencumbered access to high quality data through the emergence of open science initiatives in the historically challenging area of early stage drug discovery. At the same time, many patient-centric organisations are taking matters into their own hands by participating in, enabling and funding research. Here we present the rationale behind the innovative partnership between the Structural Genomics Consortium (SGC)-an open, pre-competitive pre-clinical research consortium and the research-focused patient organisation Myeloma UK to create a new, comprehensive platform to accelerate the discovery and development of new treatments for multiple myeloma.

BACKGROUND: Cytosine arabinoside (Ara C) is a useful chemotherapy agent, used for treating acute myeloid leukaemia, although it may be associated with side effects including painful neuropathy. It is also used for in vitro neuronal studies to limit the proliferation of non-neuronal cells and thereby select nondividing neuronal cells. We studied the effects of Ara C on human dorsal root ganglion (DRG) neurons, especially the expression and sensitivity of the ion channel TRPV1, which responds to noxious heat and capsaicin and is a key mediator of neuropathic pain. METHODS: Human DRG neurons were cultured with or without Ara C for 2 weeks, after which Ara C was discontinued. Double immunostaining for the regenerative neuronal marker Gap43 and the capsaicin receptor TRPV1 showed that the normal membrane-bound localisation of TRPV1 was absent in neurons with Ara C treatment, and as expected there was massive diminution of dividing non-neuronal cells. Calcium imaging studies showed that during exposure to Ara C the neurons lost responsiveness to capsaicin, although ionomycin responses were intact, indicating general cell viability and responsiveness. Between 2 days and up to 3 weeks after removal of Ara C, the neuronal responses to capsaicin were regained and were observed to be four times (P = 0.0008, Student's t-test) that of controls, but there was only a gradual recovery of non-neuronal cells. Three to six weeks after Ara C removal, capsaicin responses were comparable to controls. CONCLUSIONS: It is postulated that Ara C treatment blocked insertion of TRPV1 in the cell membrane, resulting in accumulation of the receptors in the cytoplasm, loss of capsaicin sensitivity, and membrane-bound immunostaining, which was restored with a rebound on withdrawal of Ara C. The observed pattern of loss of capsaicin sensitivity, followed by hypersensitivity and recovery, appears to reflect some of the features observed in chemotherapy-induced neuropathy, and may provide a model for developing new treatments and prophylaxis.

Background Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood. Methods Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF). Results TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] – no pain group, n = 8, 0.69 [0–1.27]; pain group, n = 10, 2.15 [0.77–4.38]; p = 0.0009). Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] – no pain group, n = 6, 0.75 [0–2]; pain group, n = 11, 2 123, p = 0.008; TRPV4, median [range] – no pain group, n = 6, [0–1]; pain group, n = 11, 1 [0.5–2], p = 0.014). Conclusion Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study.

Cell-attached patch recordings were made from zona-free mouse eggs to examine currents at the single-channel level; two types were identified. A channel with a mean conductance of 130 pS showed changes in reversal potential on dilution of the patch pipette solution consistent with a cation channel; the channel current persisted when the only cation in the pipette was K+: it was concluded that this channel was permeable to K+. The binomial distribution was used to describe the proportion of time 0, 1, 2, ..., n channels were open in multichannel recordings of the K+ channel. Bursts of inward currents of about 1 pA, which showed voltage activation and could be recorded in the presence of CsCl and tetraethylammonium (TEA), were identified as Ca2+ channels.

The effect of intracellular and extracellular pH on GABA-gated Cl- conductance was studied using H(+)-selective microelectrodes and a three-microelectrode voltage clamp in crayfish leg opener muscle fibres in bicarbonate-free solutions. Experimental variation of intracellular pH in the range 6.4-8.0 did not affect the GABA-gated conductance. In contrast to this, the GABA-gated conductance was sensitive to changes in external pH. Raising the external pH from 7.4 to 8.4 decreased the GABA-gated peak conductance observed immediately following application of GABA by 30%, and a change from 7.4 to 6.4 produced an increase of 26%. The effect of extracellular pH on the GABA-gated peak conductance was approximately linear in the pH range 6.4-8.9. A slight decrease in the slope of the pH-conductance relationship was evident in the pH range 5.4-6.4. The desensitization of the GABA-gated conductance was also affected by external pH. At pH 6.9 the conductance produced by 1 mM GABA showed a desensitization of about 15%, and at pH 8.9 this value was 34%. Raising the external pH in the presence of GABA decreased the GABA-gated peak conductance and increased the fractional desensitization, while lowering the external pH produced opposite effects, and was capable of repriming the conductance from a desensitized state to the non-desensitized state. The above results show that the GABA-gated conductance is sensitive to changes in external pH in the physiological range, and suggest that pH-dependent changes in the postsynaptic efficacy of GABA-mediated inhibition may contribute to H+ modulation of neuronal excitability.

1. Intracellular pH was recorded (double-barrelled pH-selective microelectrodes) in single ventricular myocytes and whole papillary muscles isolated from guinea-pig heart. Both preparations were acid-loaded by various manoeuvres (addition and removal of external NH4Cl or CO2) in order that a comparison could be made of the size and speed of intracellular pH changes and hence of the apparent intracellular buffering power (beta). 2. For the same acid-loading procedure, the size of intracellular pH (pHi) changes was about threefold larger in the isolated myocyte than in whole papillary muscle. The rate of initial acid loading as well as the subsequent rate of pHi recovery (caused by acid extrusion from the cell) were also threefold faster in the myocyte. Estimates of apparent intrinsic (non-CO2) buffering power, based upon the size of pHi changes during acid loading, were 15-20 mmol l-1 for the myocyte and about 70 mmol l-1 for whole muscle. This latter value is similar to previous estimates of beta in heart. 3. When acid extrusion was reduced by applying a high dose of amiloride (1 mmol l-1), then the size of the pHi change during acid loading increased greatly in papillary muscle but changed much less in the myocyte; beta now appeared to be about 30 mmol l-1 in whole muscle but remained essentially unchanged in the myocyte. 4. We conclude that previous values for beta in cardiac muscle have been greatly overestimated because of the presence of sarcolemmal acid extrusion. Paradoxically, this error in estimating beta is far less evident in the isolated myocyte. We suggest that this is because a much more rapid acid loading is achievable in the myocyte so that acid loading will be blunted less by acid extrusion than in whole muscle. We present a simple mathematical model that demonstrates this phenomenon. We conclude that beta in ventricular muscle is likely to resemble that measured in the isolated myocyte, i.e. 15-20 mmol l-1. 5. Slow acid loading in whole ventricular muscle will also affect the kinetics of pHi changes. The model indicates that the rate of pHi recovery from an acid load in papillary muscle does not reflect the pHi dependence of acid extrusion. Instead, it is heavily influenced by the slow rate of acid loading. This emphasises that great care should be taken when interpreting the kinetics of pHi changes in multicellular ventricular preparations.

1. The influence of repetitive activity upon intracellular pH (pHi), intracellular Na+ activity (aNA(i)) and contraction was examined in isolated sheep cardiac Purkinje fibres. Ion-selective microelectrodes were used to measure intracellular Na+ and H+ ion activity. Twitch tension was elicited by field stimulation or by depolarizing pulses applied using a two-microelectrode voltage clamp. Experiments were performed in HEPES-buffered solution equilibrated either with air or 100% O2. 2. An increase in action potential frequency from a basal rate of 0.1 to 1-4 Hz induced a reversible fall in pHi and a reversible rise in aNa(i). These effects reached a steady state 3-10 min following an increase in stimulation frequency, and showed a linear dependence on frequency with a mean slope of 0.023 pH units Hz-1 and 0.57 mmol l-1 Hz-1, respectively. The rise in total intracellular acid and aNa(i) associated with a single action potential was estimated as 5.3 mu equiv l-1 of acid and 3.5 mu equiv l-1 of Na+. 3. At action potential frequencies greater than 1 Hz, the rate-dependent rise in aNa(i) was usually accompanied by a positive force staircase. 4. The fall in pHi following a rate increase also occurred when fibres were bathed in Tyrode solution equilibrated with 23 mM-HCO3- plus nominally 5% CO2/95% O2. In these cases, however, the fall in pHi was halved in magnitude. 5. In fibres exposed to strophanthidin (0.5 microM), the rate-dependent fall in pHi was doubled in magnitude and its time course was more variable than under drug-free conditions. The rate-dependent rise in aiNa was also usually larger in strophanthidin. 6. In order to examine the influence of the rate-dependent acidosis on developed tension, the acidosis was reversed experimentally by adding 2 mmol l-1 NH4Cl to the bathing solution. This produced a rise in pHi accompanied by a large increase in twitch tension. Such an effect of pHi upon tension was quantitatively similar to that observed in previous work on Purkinje fibres (Vaughan-Jones, Eisner and Lederer, 1987). 7. It is concluded that the rate dependence of pHi will influence both the magnitude and the time course of an inotropic response to a change in heart rate.

Measurements of membrane potential and resistance have been made in zona-free eggs of mice and hamsters. The mean +/- S.D. values for membrane potential were -91 +/- 28 mV (mouse) and -97 +/- 29 mV (hamster) and for input resistance were 430 +/- 230 M omega (mouse) and 410 +/- 150 M omega (hamster) respectively. Large fluctuations (20 mV) of membrane potential occurred apparently at random and these were accompanied by changes of membrane resistance. Depolarizing current pulses passed through the recording micro-electrode evoked action potentials in eggs of both species. The threshold for excitation was about -50 mV, the maximum rate of rise of the action potential was about 3 V.s-1 and its peak value was about +13 mV. Action potentials could be evoked in eggs bathed in sodium-free solution or in normal solution containing tetrodotoxin (3 microM). The presence of cobalt (5-20 mM), lanthanum (1 mM) or verapamil (200-400 microM) in the bathing solution suppressed the action potential. Raising the extracellular calcium concentration from 4 to 40 mM increased the peak value of the action potential by 25 mV. It is concluded that the plasma membranes of mouse and hamster eggs have voltage-dependent calcium channels.

Background The Bladder cooling reflex (BCR) i.e. uninhibited detrusor contractions evoked by intravesical instillation of cold saline, is a segmental reflex believed to be triggered by menthol sensitive cold receptors in the bladder wall, with the afferent signals transmitted by C fibres. The BCR is a neonatal reflex that becomes suppressed by descending signals from higher centres at approximately the time when the child gains full voluntary control of voiding. It re-emerges in adults with neurogenic detrusor overactivity as a consequence of loss of central descending inhibition, resulting from conditions such as spinal cord injury or multiple sclerosis. We have recently shown an increase of nerve fibres expressing the cool and menthol receptor TRPM8 in both overactive (IDO) and painful bladder syndrome (PBS), but its functional significance is unknown. We have therefore studied the bladder cooling reflex and associated sensory symptoms in patients with PBS and overactivity disorders. Methods The BCR, elicited by ice water test (IWT) was performed in patients with painful bladder syndrome (PBS, n = 17), idiopathic detrusor overactivity (IDO, n = 22), neurogenic detrusor overactivity (NDO, n = 4) and stress urinary incontinence (as controls, n = 21). The IWT was performed by intravesical instillation of cold saline (0 – 4°C). A positive IWT was defined as presence of uninhibited detrusor contraction evoked by cold saline, associated with urgency or with fluid expulsion. Patients were asked to report and rate any pain and cold sensation during the test. Results A positive IWT was observed in IDO (6/22, 27.3%) and NDO (4/4, 100%) patients, but was negative in all control and PBS patients. Thirteen (76.5%) PBS patients reported pain during the IWT, with significantly higher pain scores during ice water instillation compared to the baseline (P = 0.0002), or equivalent amount of bladder filling (100 mls) with saline at room temperature (P = 0.015). None of the control or overactive (NDO/IDO) patients reported any pain during the IWT. Conclusion The BCR in DO may reflect loss of central inhibition, which appears necessary to elicit this reflex; the pain elicited in PBS suggests afferent sensitisation, hence sensory symptoms are evoked but not reflex detrusor contractions. The ice water test may be a useful and simple marker for clinical trials in PBS, particularly for novel selective TRPM8 antagonists.

Industry, government, patient advocacy groups, public funders, and academic thought leaders met in Toronto, Canada, to set into motion an initiative that addresses some of the scientific and organizational challenges of modern therapeutics discovery. What emerged from the meeting was a public-private partnership that seeks to establish proof of clinical mechanism (POCM) for selected "pioneer" disease targets using lead compounds-all accomplished in the precompetitive space. The group will reconvene in April 2011 to create a business plan that specifies the generation of two positive POCM results per year.

Mechanosensitive ion channels convert external mechanical force into electrical and chemical signals in cells, but their physiological function in different tissues is not clearly understood. One reason for this is that there is as yet no satisfactory physiological method to stimulate these channels in living cells. Using the nanopipette-probe of the Scanning Ion Conductance Microscope (SICM), we have developed a new technique to apply local mechanical stimulus to living cells to an area of about 0.385 microm2, determined by the pipette diameter. Our method prevents any physical contact and damage to the cell membrane by use of a pressure jet applied via the nanopipette. The study used whole-cell patch-clamp recordings and measurements of intracellular Ca2+ concentration to validate the application of the mechanical stimulation protocols in human and rat dorsal root ganglia (DRG) sensory neurons. We were able, for the first time, to produce a non-contact, controlled mechanical stimulation on living neurites of human DRG neurons. Our methods will enable the identification and characterisation of compounds being developed for the treatment of clinical mechanical hypersensitivity states.

The objective measurement of chronic pain in pre-clinical animal models and in the clinical arena is problematic. The multifactorial nature of pain can adversely affect the accuracy with which it can be quantitatively assessed. This is due to diverse aetiology, environmental factors and psychological status which can significantly affect the measurement of pain in animal models and presentation in the clinic. In pre-clinical studies, the objectivity of pain measurement is affected by variability in behavioural phenotype and the lack of verbal description, while clinical pain is subject to variance by a considerable placebo effect, thought to be related to merely being in a clinical setting. Therefore, it would be advantageous to identify a biological marker which is sensitive to pain pathology and modulated by an efficacious analgesic/anti-inflammatory agent. The present study highlights several metabolites present in urine that are modulated from basal levels by pain and inflammation induced following hindpaw injection of Freund's Complete Adjuvant. Nuclear magnetic resonance (NMR) analysis of urine and multivariate statistical data analysis (MVDA) were used to examine in detail the modulation of small molecule candidate biomarkers or surrogates. Several molecules were shown by NMR to be modulated by FCA injection including carboxylates, acetylated metabolites, choline metabolites, tricarboxylic acid cycle intermediates, creatine and creatinine, taurine, N-methylnicotinamide and its metabolites and several unassigned peaks. © Springer Science+Business Media, LLC 2006.

The cannabinoid agonist, HU210 has been evaluated in vivo in nociceptive and inflammatory pain models in the rat. The ED50 for the anti-nociceptive (increasing mechanical withdrawal threshold) effect was 0.1 mg/kg-1 i.p., and for anti-hypersensitivity and anti-inflammatory activity was 5 g/kg-1 i.p. (in the carrageenan model). The selective CB1 antagonist, AM281 (0.5 microg/kg-1 i.p.) reversed effects of HU210 (10 and 30 microg/kg-1 i.p.) in both nociceptive and inflammatory models of hypersensitivity. The selective CB2 antagonist, SR144528 (1 mg/kg-1 i.p.) antagonised effects of HU210 (30 microg/kg-1 i.p.) in the carrageenan induced inflammatory hypersensitivity. The CB2 agonist, 1-(2,3-Dichlorobenzoyl)-5-methoxy-2-methyl-(2-(morpholin-4-yl)ethyl)-1H-indole (GW405833) inhibited the hypersensitivity and was anti-inflammatory in vivo. These effects were blocked by SR144528. These findings suggest that CB1 receptors are involved in nociceptive pain and that both CB1 and CB2 receptors are involved in inflammatory hypersensitivity. Future studies will investigate effects on identified inflammatory cells within the inflamed tissue to further elucidate the role of cannabinoid receptors.

Chronic constriction injury (CCI) of the sciatic nerve results in persistent mechanical hyperalgesia together with Fos protein expression in the lumbar spinal cord. We have examined the relationship between mechanical hyperalgesia and Fos expression within the lumbar spinal cord on days 14, 35 and 55 after either CCI or sham operation. To determine the role of NMDA receptor mechanisms in the maintenance of hyperalgesia and Fos expression, the NMDA antagonist MK-801 (0.3 mg kg-1 s.c.) was administered daily on days 28 to 34 after operation. CCI animals developed unilateral hind limb hyperalgesia that persisted unchanged from days 14 to 55 of the study. MK-801 treatment reduced hyperalgesia by 57% (p=0.02) on day 35 in CCI animals but did influence hyperalgesia at day 55. In the spinal cord, Fos positive cells were present bilaterally throughout laminae 3-10 at all time points examined in both CCI and sham group animals. Fos counts ipsilateral to the side of injury in laminae 3-10 correlated significantly with hyperalgesia scores in the CCI but not sham animals. MK-801 treatment resulted in a suppression of Fos expression in ipsilateral laminae 3-4 (p=0.0017) and laminae 5-10 (p=0.0026) of CCI animals on day 35. Fos expression in sham group animals was not inhibited by MK-801 treatment at day 35. These results indicate that Fos expression is maintained by differing mechanisms following nerve injury or sham operation. The functional consequences of Fos expression following nerve injury and sham operation are discussed.

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Sensory neurones co-express voltage-gated sodium channels that mediate TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents, which may contribute to chronic pain after nerve injury. We previously demonstrated that TTX-R channels were decreased acutely in human sensory cell bodies after central axotomy, but accumulated in nerve terminals after peripheral axotomy. We have now studied the TTX-S channels PN I and Brain III, using specific antibodies for immunohistochemistry, in dorsal root ganglia (DRG) from 10 patients with traumatic central axotomy, nerves from 16 patients with peripheral axotomy, and controls. PN1 showed temporal changes similar to the TTX-R channels in sensory cell bodies of injured DRG. In contrast, Brain III was found only in injured nerves (not control nerves, or control/central axotomy DRG). PN1 and Brain III are distinct targets for novel analgesics.

1. Following our earlier observations that the tachykinin NK1 receptor antagonist CP-99,994 is an effective anti-emetic in ferrets, we have examined the anti-emetic effects of a more potent and novel NK1 receptor antagonist, GR203040, against various emetic stimuli in the ferret, dog and house musk shrew (Suncus murinus). 2. In ferrets, GR203040 (0.1 mg kg-1 s.c. or i.v.) is effective against emesis induced by radiation, cisplatin, cyclophosphamide, copper sulphate, ipecacuanha or morphine. 3. In animals in which emesis had been established with cisplatin, GR203040 (1 mg kg-1 s.c.) was fully effective as an interventional treatment. No further emesis was seen in animals treated with GR203040 whilst saline-treated animals continued to vomit. 4. GR203040 (0.1 mg kg-1 s.c.) retains anti-emetic efficacy in the ferret, even when given as a 6 h pretreatment, indicating that this compound has a long duration of action. The compound is also effective orally at the same dose, when given as a 90 min pretreatment. 5. GR203040 (0.1 mg kg-1 i.v.) is fully effective against ipecacuanha-induced emesis in the dog. 6. GR203040 is effective against motion- and cisplatin-induced emesis in Suncus murinus. These effects were seen at doses an order of magnitude greater than those shown to be effective against cisplatin in the ferret. 7. In conclusion, GR203040 is a novel anti-emetic agent, and the broad spectrum of anti-emetic activity, together with activity observed in three species, suggests that this compound is worthy of clinical investigation.

Functional gastrointestinal disorders, such as irritable bowel syndrome and functional dyspepsia, are complex conditions with multiple factors contributing to their pathophysiology. As a consequence they are difficult to treat and have posed significant challenges to the pharmaceutical industry when trying to develop new and effective treatments. This review provides an overview of these difficulties and how the industry is reshaping its drug developmental strategies. It describes some of the more significant and encouraging advances that have occurred, and discusses how future research might embrace the opportunities provided by advances in genetic and in particular, epigenetic research.

Measurements have been made of membrane potential and input resistance of zona-free hamster eggs from single micro-electrode recordings. At room temperature (20-23 degrees C) the mean (+/- S.D.) values for the potential and resistance were -30 +/- 8 mV and 280 +/- 130 M omega (n = 94 eggs). At 37 degrees C the mean (+/- S.D.) values for the potential and resistance were -39 +/- 13 mV and 230 +/- 60 M omega (n = 60 eggs). The most negative potential recorded at room temperature was -51 mV in a cell which had an input resistance of 620 M omega. At 37 degrees C six eggs out of sixty had potentials more negative than -50 mV and three of these gave all-or-none action potentials in response to depolarizing current pulses. In a separate series of experiments with high resistance micro-electrodes (ca. 100 M omega) six eggs out of twenty-one had potentials more negative than -50 mV and four of these were electrically excitable. Transient potential recordings during impalement indicated that the potential was more negative than -30 mV but that the insertion of a micro-electrode produced a leak pathway with a resistance of about 10 M omega, substantially smaller than the steady-state estimates of the input resistance (see above). Whole-cell recordings with patch pipettes gave potentials in the range -30 to -80 mV and input resistances in the range 180 to 350 M omega (n = 8); four eggs gave action potentials in response to depolarizing current pulses passed through the patch pipette. It is concluded that the leak impalement artifact is so significant in micro-electrode recordings from hamster eggs that it prevents routine reliable potential measurements.

Background: The angiotensin II (AngII) receptor subtype 2 (AT2R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. Methods: We used immunostaining with characterized antibodies to study the localization of AT2R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. Results: AT2R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1R antagonist losartan had no effect on capsaicin responses. AT2R was localized in sensoryneuronsofhumanDRG,andnervefibresinperipheralnerves,skin, urinary bladder and bowel. A majority sub-population (60%) of small-/ medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. Conclusions: AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.

Epigenetic regulation of gene expression is a dynamic and reversible process that establishes normal cellular phenotypes but also contributes to human diseases. At the molecular level, epigenetic regulation involves hierarchical covalent modification of DNA and the proteins that package DNA, such as histones. Here, we review the key protein families that mediate epigenetic signalling through the acetylation and methylation of histones, including histone deacetylases, protein methyltransferases, lysine demethylases, bromodomain-containing proteins and proteins that bind to methylated histones. These protein families are emerging as druggable classes of enzymes and druggable classes of proteing-protein interaction domains. In this article, we discuss the known links with disease, basic molecular mechanisms of action and recent progress in the pharmacological modulation of each class of proteins. © 2012 Macmillan Publishers Limited. All rights reserved.