TDI Publications 2016

Smida M, Fece de la Cruz F, Kerzendorfer C, Uras IZ, Mair B, Mazouzi A, Suchankova T, Konopka T, Katz AM, Paz K et al. 2016. MEK inhibitors block growth of lung tumours with mutations in ataxia-telangiectasia mutated. Nat Commun, 7 (1), pp. 13701. | Citations: 10 (Scopus) | Show Abstract | Read more

Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.

New M, Sheikh S, Bekheet M, Olzscha H, Thezenas M-L, Care MA, Fotheringham S, Tooze RM, Kessler B, La Thangue NB. 2016. TLR Adaptor Protein MYD88 Mediates Sensitivity to HDAC Inhibitors via a Cytokine-Dependent Mechanism. Cancer Res, 76 (23), pp. 6975-6987. | Citations: 5 (Web of Science Lite) | Show Abstract | Read more

Histone deacetylase (HDAC) inhibitors have proven useful therapeutic agents for certain hematologic cancers. However, HDAC inhibition causes diverse cellular outcomes, and identification of cancer-relevant pathways within these outcomes remains unresolved. In this study, we utilized an unbiased loss-of-function screen and identified the Toll-like receptor (TLR) adaptor protein MYD88 as a key regulator of the antiproliferative effects of HDAC inhibition. High expression of MYD88 exhibited increased sensitivity to HDAC inhibitors; conversely, low expression coincided with reduced sensitivity. MYD88-dependent TLR signaling controlled cytokine levels, which then acted via an extracellular mechanism to maintain cell proliferation and sensitize cells to HDAC inhibition. MYD88 activity was directly regulated through lysine acetylation and was deacetylated by HDAC6. MYD88 was a component of a wider acetylation signature in the ABC subgroup of diffuse large B-cell lymphoma, and one of the most frequent mutations in MYD88, L265P, conferred increased cell sensitivity to HDAC inhibitors. Our study defines acetylation of MYD88, which, by regulating TLR-dependent signaling to cytokine genes, influences the antiproliferative effects of HDAC inhibitors. Our results provide a possible explanation for the sensitivity of malignancies of hematologic origin to HDAC inhibitor-based therapy. Cancer Res; 76(23); 6975-87. ©2016 AACR.

Martins R, Maier J, Gorki A-D, Huber KVM, Sharif O, Starkl P, Saluzzo S, Quattrone F, Gawish R, Lakovits K et al. 2016. Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions. Nat Immunol, 17 (12), pp. 1361-1372. | Citations: 24 (Scopus) | Show Abstract | Read more

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

Chaikuad A, Diharce J, Schröder M, Foucourt A, Leblond B, Casagrande A-S, Désiré L, Bonnet P, Knapp S, Besson T. 2016. An Unusual Binding Model of the Methyl 9-Anilinothiazolo[5,4-f] quinazoline-2-carbimidates (EHT 1610 and EHT 5372) Confers High Selectivity for Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases. J Med Chem, 59 (22), pp. 10315-10321. | Citations: 10 (Web of Science Lite) | Show Abstract | Read more

Methyl 9-anilinothiazolo[5,4-f]quinazoline-2-carbimidates 1 (EHT 5372) and 2 (EHT 1610) are strong inhibitors of DYRK's family kinases. The crystal structures of the complex revealed a noncanonical binding mode of compounds 1 and 2 in DYRK2, explaining the remarkable selectivity and potency of these inhibitors. The structural data and comparison presented here provide therefore a template for further improvement of this inhibitor class and for the development of novel inhibitors selectively targeting DYRK kinases.

Vaz B, Popovic M, Newman JA, Fielden J, Aitkenhead H, Halder S, Singh AN, Vendrell I, Fischer R, Torrecilla I et al. 2016. Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair. Mol Cell, 64 (4), pp. 704-719. | Citations: 39 (Scopus) | Show Abstract | Read more

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

Forster M, Chaikuad A, Bauer SM, Holstein J, Robers MB, Corona CR, Gehringer M, Pfaffenrot E, Ghoreschi K, Knapp S, Laufer SA. 2016. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem Biol, 23 (11), pp. 1335-1340. | Citations: 18 (Scopus) | Show Abstract | Read more

Janus kinases (JAKs) are a family of cytoplasmatic tyrosine kinases that are attractive targets for the development of anti-inflammatory drugs given their roles in cytokine signaling. One question regarding JAKs and their inhibitors that remains under intensive debate is whether JAK inhibitors should be isoform selective. Since JAK3 functions are restricted to immune cells, an isoform-selective inhibitor for JAK3 could be especially valuable to achieve clinically more useful and precise effects. However, the high degree of structural conservation makes isoform-selective targeting a challenging task. Here, we present picomolar inhibitors with unprecedented kinome-wide selectivity for JAK3. Selectivity was achieved by concurrent covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. We confirmed that in vitro activity and selectivity translate well into the cellular environment and suggest that our inhibitors are powerful tools to elucidate JAK3-specific functions.

Wright TH, Bower BJ, Chalker JM, Bernardes GJL, Wiewiora R, Ng W-L, Raj R, Faulkner S, Vallée MRJ, Phanumartwiwath A et al. 2016. Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity. Science, 354 (6312), pp. aag1465-aag1465. | Citations: 62 (Web of Science Lite) | Show Abstract | Read more

Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp3)-C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cβ-Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

Horvath P, Aulner N, Bickle M, Davies AM, Nery ED, Ebner D, Montoya MC, Östling P, Pietiäinen V, Price LS et al. 2016. Screening out irrelevant cell-based models of disease. Nat Rev Drug Discov, 15 (11), pp. 751-769. | Citations: 114 (Scopus) | Show Abstract | Read more

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

Keating SM, Heitman JW, Wu S, Deng X, Stacey AR, Zahn RC, de la Rosa M, Finstad SL, Lifson JD, Piatak M et al. 2016. Magnitude and Quality of Cytokine and Chemokine Storm during Acute Infection Distinguish Nonprogressive and Progressive Simian Immunodeficiency Virus Infections of Nonhuman Primates. J Virol, 90 (22), pp. 10339-10350. | Citations: 4 (Web of Science Lite) | Show Abstract | Read more

Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δnef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δnef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression. IMPORTANCE: NHP models of HIV infection constitute a powerful tool with which to study viral pathogenesis in order to gain critical information for a better understanding of HIV infection in humans. Here we studied progressive and nonprogressive simian immunodeficiency virus (SIV) infection models in both natural and nonnatural host NHP species. Regardless of the pathogenicity of the virus infection and regardless of the NHP species studied, the magnitude of viremia, as measured by area under the curve, during the first 4 weeks of infection correlated positively with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between nonprogressive and progressive SIV infection models. The qualitative differences in the early immune response in pathogenic and nonpathogenic infections identified here may be important determinants of the subsequent disease course.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2016. Restoring p53 Function in Human Melanoma Cells by Inhibiting MDM2 and Cyclin B1/CDK1-Phosphorylated Nuclear iASPP. Cancer Cell, 30 (5), pp. 822-823. | Citations: 7 (Web of Science Lite) | Show Abstract | Read more

© 2016 Elsevier Inc. (Cancer Cell 23, 618–633; May 13, 2013) In the original Figure 4G, two of the upper panels (His-iASPP(625-828)-FITC; the two rightmost panels) were inadvertently duplicated in the lower set of panels (His-ASPP2(905-1128)-FITC; third and fourth from left). This was a mistake made by the authors during the assembly of the figure. This error does not affect any of the findings reported in the paper. The corrected Figure 4 is presented below. The authors apologize for any confusion that this error may have caused. [figure presented]

Ding S, Medjahed H, Prévost J, Coutu M, Xiang S-H, Finzi A. 2016. Lineage-Specific Differences between the gp120 Inner Domain Layer 3 of Human Immunodeficiency Virus and That of Simian Immunodeficiency Virus. J Virol, 90 (22), pp. 10065-10073. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

Binding of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological layers (layer 1, layer 2, and layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to layer 1 of HIV-1 gp120, the SIVmac239 gp120 layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved tryptophan at position 375 (Trp 375) in HIV-2/SIVsmm. In this study, we investigated the role of SIV layer 3 in viral entry, cell-to-cell fusion, and CD4 binding. We observed that a network of interactions involving some residues of the β8-α5 region in SIVmac239 layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity, which directly contacts CD4. In summary, our results suggest that layer 3 in SIV has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in layer 3 from HIV-1 and that from SIV. IMPORTANCE: CD4-induced conformational changes in the gp120 inner domain involve rearrangements between three topological layers. While the role of layers 1 to 3 for HIV-1 and layers 1 and 2 for SIV on gp120 transition to the CD4-bound conformation has been reported, the role of SIV layer 3 remains unknown. Here we report that SIV layer 3 has a greater impact on CD4 binding than does layer 3 in HIV-1 gp120. This work defines lineage-specific differences in layer 3 from HIV-1 and SIV.

Grampp S, Platt JL, Lauer V, Salama R, Kranz F, Neumann VK, Wach S, Stöhr C, Hartmann A, Eckardt K-U et al. 2016. Genetic variation at the 8q24.21 renal cancer susceptibility locus affects HIF binding to a MYC enhancer. Nat Commun, 7 (1), pp. 13183. | Citations: 17 (Web of Science Lite) | Show Abstract | Read more

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of function of the von Hippel-Lindau tumour suppressor (VHL) and unrestrained activation of hypoxia-inducible transcription factors (HIFs). Genetic and epigenetic determinants have an impact on HIF pathways. A recent genome-wide association study on renal cancer susceptibility identified single-nucleotide polymorphisms (SNPs) in an intergenic region located between the oncogenes MYC and PVT1. Here using assays of chromatin conformation, allele-specific chromatin immunoprecipitation and genome editing, we show that HIF binding to this regulatory element is necessary to trans-activate MYC and PVT1 expression specifically in cells of renal tubular origins. Moreover, we demonstrate that the risk-associated polymorphisms increase chromatin accessibility and activity as well as HIF binding to the enhancer. These findings provide further evidence that genetic variation at HIF-binding sites modulates the oncogenic transcriptional output of the VHL-HIF axis and provide a functional explanation for the disease-associated effects of SNPs in ccRCC.

Gebel J, Luh LM, Coutandin D, Osterburg C, Löhr F, Schäfer B, Frombach A-S, Sumyk M, Buchner L, Krojer T et al. 2016. Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization. Cell Death Differ, 23 (12), pp. 1930-1940. | Citations: 13 (Web of Science Lite) | Show Abstract | Read more

Members of the p53 tumor-suppressor family are expressed as multiple isoforms. Isoforms with an N-terminal transactivation domain are transcriptionally active, while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumor-suppressor function of TAp73β. This can in principle be due to blocking of the promoter or by direct interaction between both proteins. p63 and p73 can hetero-oligomerize through their tetramerization domains and a hetero-tetramer consisting of two p63 and two p73 molecules is thermodynamically more stable than both homo-tetramers. Here we show that cells expressing both p63 and p73 exist in mouse epidermis and hair follicle and that hetero-tetramer complexes can be detected by immunoprecipitation in differentiating keratinocytes. Through structure determination of the hetero-tetramer, we reveal why this hetero-tetramer is the thermodynamically preferred species. We have created mutants that exclusively form either hetero-tetramers or homo-tetramers, allowing to investigate the function of these p63/p73 hetero-tetramers. Using these tools, we show that inhibition of TAp73β in squamous cell carcinomas is due to promoter squelching and not direct interaction.

Rose NR, King HW, Blackledge NP, Fursova NA, Ember KJ, Fischer R, Kessler BM, Klose RJ. 2016. RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes. Elife, 5 | Citations: 12 (Web of Science Lite) | Show Abstract | Read more

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation.

Picaud S, Leonards K, Lambert J-P, Dovey O, Wells C, Fedorov O, Monteiro O, Fujisawa T, Wang C-Y, Lingard H et al. 2016. Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia. Sci Adv, 2 (10), pp. e1600760. | Citations: 19 (Scopus) | Show Abstract | Read more

Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.

Welker F, Hajdinjak M, Talamo S, Jaouen K, Dannemann M, David F, Julien M, Meyer M, Kelso J, Barnes I et al. 2016. Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne. Proc Natl Acad Sci U S A, 113 (40), pp. 11162-11167. | Citations: 38 (Scopus) | Show Abstract | Read more

In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this "transitional" assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal-Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne.

Myrianthopoulos V, Gaboriaud-Kolar N, Tallant C, Hall M-L, Grigoriou S, Brownlee PM, Fedorov O, Rogers C, Heidenreich D, Wanior M et al. 2016. Discovery and Optimization of a Selective Ligand for the Switch/Sucrose Nonfermenting-Related Bromodomains of Polybromo Protein-1 by the Use of Virtual Screening and Hydration Analysis. J Med Chem, 59 (19), pp. 8787-8803. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein-ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5).

Demarchi B, Hall S, Roncal-Herrero T, Freeman CL, Woolley J, Crisp MK, Wilson J, Fotakis A, Fischer R, Kessler BM et al. 2016. Protein sequences bound to mineral surfaces persist into deep time. Elife, 5 (September), | Citations: 37 (Scopus) | Show Abstract | Read more

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C).

Popp TA, Tallant C, Rogers C, Fedorov O, Brennan PE, Müller S, Knapp S, Bracher F. 2016. Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors. J Med Chem, 59 (19), pp. 8889-8912. | Citations: 20 (Web of Science Lite) | Show Abstract | Read more

CBP (CREB (cAMP responsive element binding protein) binding protein (CREBBP)) and P300 (adenovirus E1A-associated 300 kDa protein) are two closely related histone acetyltransferases (HATs) that play a key role in the regulation of gene transcription. Both proteins contain a bromodomain flanking the HAT catalytic domain that is important for the targeting of CBP/P300 to chromatin and which offeres an opportunity for the development of protein-protein interaction inhibitors. Here we present the development of CBP/P300 bromodomain inhibitors with 2,3,4,5-tetrahydro-1,4-benzoxazepine backbone, an N-acetyl-lysine mimetic scaffold that led to the recent development of the chemical probe I-CBP112. We present comprehensive SAR of this inhibitor class as well as demonstration of cellular on target activity of the most potent and selective inhibitor TPOP146, which showed 134 nM affinity for CBP with excellent selectivity over other bromodomains.

Kaelin WG, Ratcliffe PJ, Semenza GL. 2016. Pathways for Oxygen Regulation and Homeostasis JAMA, 316 (12), pp. 1252-1252. | Read more

Kaisar M, van Dullemen LFA, Thézénas M-L, Zeeshan Akhtar M, Huang H, Rendel S, Charles PD, Fischer R, Ploeg RJ, Kessler BM. 2016. Plasma degradome affected by variable storage of human blood. Clin Proteomics, 13 (1), pp. 26. | Citations: 7 (Scopus) | Show Abstract | Read more

BACKGROUND: The successful application of-omics technologies in the discovery of novel biomarkers and targets of therapeutic interventions is facilitated by large collections of well curated clinical samples stored in bio banks. Mining the plasma proteome holds promise to improve our understanding of disease mechanisms and may represent a source of biomarkers. However, a major confounding factor for defining disease-specific proteomic signatures in plasma is the variation in handling and processing of clinical samples leading to protein degradation. To address this, we defined a plasma proteolytic signature (degradome) reflecting pre-analytical variability in blood samples that remained at ambient temperature for different time periods after collection and prior to processing. METHODS: We obtained EDTA blood samples from five healthy volunteers (n = 5), and blood tubes remained at ambient temperature for 30 min, 8, 24 and 48 h prior to centrifugation and isolation of plasma. Naturally occurred peptides derived from plasma samples were compared by label-free quantitative LC-MS/MS. To profile protein degradation, we analysed pooled plasma samples at T = 30 min and 48 h using PROTOMAP analysis. The proteolytic pattern of selected protein candidates was further validated by immunoblotting. RESULTS: A total of 820 plasma proteins were surveyed by PROTOMAP, and for 4 % of these, marked degradation was observed. We show distinct proteolysis patterns for talin-1, coagulation factor XI, complement protein C1r, C3, C4 and thrombospondin, and several proteins including S100A8, A9, annexin A1, profiling-1 and platelet glycoprotein V are enriched after 48 h blood storage at ambient temperature. In particular, thrombospondin protein levels increased after 8 h and proteolytic fragments appeared after 24 h storage time. CONCLUSIONS: The overall impact of blood storage at ambient temperature for variable times on the plasma proteome and degradome is relatively minor, but in some cases can cause a potential bias in identifying and assigning relevant proteomic markers. The observed effects on the plasma proteome and degradome are predominantly triggered by limited leucocyte and platelet cell activation due to blood handling and storage. The baseline plasma degradome signature presented here can help filtering candidate protein markers relevant for clinical biomarker studies.

Bardella C, Al-Dalahmah O, Krell D, Brazauskas P, Al-Qahtani K, Tomkova M, Adam J, Serres S, Lockstone H, Freeman-Mills L et al. 2016. Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis. Cancer Cell, 30 (4), pp. 578-594. | Citations: 29 (Scopus) | Show Abstract | Read more

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

Camps C, Petousi N, Bento C, Cario H, Copley RR, McMullin MF, van Wijk R, Ratcliffe PJ, Robbins PA, Taylor JC, WGS500 Consortium. 2016. Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations. Haematologica, 101 (11), pp. 1306-1318. | Citations: 13 (Scopus) | Show Abstract | Read more

Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

Navratilova I, Aristotelous T, Picaud S, Chaikuad A, Knapp S, Filappakopoulos P, Hopkins AL. 2016. Discovery of New Bromodomain Scaffolds by Biosensor Fragment Screening. ACS Med Chem Lett, 7 (12), pp. 1213-1218. | Citations: 7 (Scopus) | Show Abstract | Read more

The discovery of novel bromodomain inhibitors by fragment screening is complicated by the presence of dimethyl sulfoxide (DMSO), an acetyl-lysine mimetic, that can compromise the detection of low affinity fragments. We demonstrate surface plasmon resonance as a primary fragment screening approach for the discovery of novel bromodomain scaffolds, by describing a protocol to overcome the DMSO interference issue. We describe the discovery of several novel small molecules scaffolds that inhibit the bromodomains PCAF, BRD4, and CREBBP, representing canonical members of three out of the seven subfamilies of bromodomains. High-resolution crystal structures of the complexes of key fragments binding to BRD4(1), CREBBP, and PCAF were determined to provide binding mode data to aid the development of potent and selective inhibitors of PCAF, CREBBP, and BRD4.

Kaelin WG, Ratcliffe PJ, Semenza GL. 2016. Pathways for Oxygen Regulation and Homeostasis: The 2016 Albert Lasker Basic Medical Research Award. JAMA, 316 (12), pp. 1252-1253. | Citations: 8 (Web of Science Lite) | Read more

Monteiro J, Kaelin W, Ratcliffe P. 2016. Making Sense of the Unexpected. Cell, 167 (1), pp. 35-38. | Show Abstract | Read more

The 2016 Albert Lasker Basic Medical Research Award is being awarded to Gregg Semenza, William Kaelin, and Peter Ratcliffe for discovery of the pathway by which human and animal cells sense and adapt to changes in oxygen availability-an essential requirement for survival. Bill and Peter joined Cell editor João Monteiro in an informal conversation about science, medicine, designing experiments, and training the next generation.

Moustakim M, Clark PGK, Hay DA, Dixon DJ, Brennan PE. 2016. Chemical probes and inhibitors of bromodomains outside the BET family. Medchemcomm, 7 (12), pp. 2246-2264. | Citations: 17 (Scopus) | Show Abstract | Read more

In the last five years, the development of inhibitors of bromodomains has emerged as an area of intensive worldwide research. Emerging evidence has implicated a number of non-BET bromodomains in the onset and progression of diseases such as cancer, HIV infection and inflammation. The development and use of small molecule chemical probes has been fundamental to pre-clinical evaluation of bromodomains as targets. Recent efforts are described highlighting the development of potent, selective and cell active non-BET bromodomain inhibitors and their therapeutic potential. Over half of typical bromodomains now have reported ligands, but those with atypical binding site residues remain resistant to chemical probe discovery efforts.

Mair B, Konopka T, Kerzendorfer C, Sleiman K, Salic S, Serra V, Muellner MK, Theodorou V, Nijman SMB. 2016. Gain- and Loss-of-Function Mutations in the Breast Cancer Gene GATA3 Result in Differential Drug Sensitivity. PLoS Genet, 12 (9), pp. e1006279. | Citations: 10 (Scopus) | Show Abstract | Read more

Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.

Thomsen SK, Ceroni A, van de Bunt M, Burrows C, Barrett A, Scharfmann R, Ebner D, McCarthy MI, Gloyn AL. 2016. Systematic Functional Characterization of Candidate Causal Genes for Type 2 Diabetes Risk Variants. Diabetes, 65 (12), pp. 3805-3811. | Citations: 27 (Scopus) | Show Abstract | Read more

Most genetic association signals for type 2 diabetes risk are located in noncoding regions of the genome, hindering translation into molecular mechanisms. Physiological studies have shown a majority of disease-associated variants to exert their effects through pancreatic islet dysfunction. Systematically characterizing the role of regional transcripts in β-cell function could identify the underlying disease-causing genes, but large-scale studies in human cellular models have previously been impractical. We developed a robust and scalable strategy based on arrayed gene silencing in the human β-cell line EndoC-βH1. In a screen of 300 positional candidates selected from 75 type 2 diabetes regions, each gene was assayed for effects on multiple disease-relevant phenotypes, including insulin secretion and cellular proliferation. We identified a total of 45 genes involved in β-cell function, pointing to possible causal mechanisms at 37 disease-associated loci. The results showed a strong enrichment for genes implicated in monogenic diabetes. Selected effects were validated in a follow-up study, including several genes (ARL15, ZMIZ1, and THADA) with previously unknown or poorly described roles in β-cell biology. We have demonstrated the feasibility of systematic functional screening in a human β-cell model and successfully prioritized plausible disease-causing genes at more than half of the regions investigated.

Elliott PR, Leske D, Hrdinka M, Bagola K, Fiil BK, McLaughlin SH, Wagstaff J, Volkmar N, Christianson JC, Kessler BM et al. 2016. SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling. Mol Cell, 63 (6), pp. 990-1005. | Citations: 40 (Scopus) | Show Abstract | Read more

The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.

Chan MC, Ilott NE, Schödel J, Sims D, Tumber A, Lippl K, Mole DR, Pugh CW, Ratcliffe PJ, Ponting CP, Schofield CJ. 2016. Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases. J Biol Chem, 291 (39), pp. 20661-20673. | Citations: 15 (Scopus) | Show Abstract | Read more

The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.

Al Haj Zen A, Nawrot DA, Howarth A, Caporali A, Ebner D, Vernet A, Schneider JE, Bhattacharya S. 2016. The Retinoid Agonist Tazarotene Promotes Angiogenesis and Wound Healing. Mol Ther, 24 (10), pp. 1745-1759. | Citations: 5 (Scopus) | Show Abstract | Read more

Therapeutic angiogenesis is a major goal of regenerative medicine, but no clinically approved small molecule exists that enhances new blood vessel formation. Here we show, using a phenotype-driven high-content imaging screen of an annotated chemical library of 1,280 bioactive small molecules, that the retinoid agonist Tazarotene, enhances in vitro angiogenesis, promoting branching morphogenesis, and tubule remodeling. The proangiogenic phenotype is mediated by retinoic acid receptor but not retinoic X receptor activation, and is characterized by secretion of the proangiogenic factors hepatocyte growth factor, vascular endothelial growth factor, plasminogen activator, urokinase and placental growth factor, and reduced secretion of the antiangiogenic factor pentraxin-3 from adjacent fibroblasts. In vivo, Tazarotene enhanced the growth of mature and functional microvessels in Matrigel implants and wound healing models, and increased blood flow. Notably, in ear punch wound healing model, Tazarotene promoted tissue repair characterized by rapid ear punch closure with normal-appearing skin containing new hair follicles, and maturing collagen fibers. Our study suggests that Tazarotene, an FDA-approved small molecule, could be potentially exploited for therapeutic applications in neovascularization and wound healing.

Nowak RP, Tumber A, Johansson C, Che KH, Brennan P, Owen D, Oppermann U. 2016. Advances and challenges in understanding histone demethylase biology. Curr Opin Chem Biol, 33 pp. 151-159. | Citations: 12 (Scopus) | Show Abstract | Read more

Within the last decade we have witnessed significant progress in the field of chromatin methylation, ranging from the discovery that chromatin methylation is reversible, to the identification of two classes of oxidative chromatin demethylases. Multiple genetic and cellular studies emphasize the role of members of the amine oxidase and 2-oxoglutarate oxygenase enzyme families involved in methyl-lysine in physiology and disease. Advances in understanding of the underlying biochemistry have resulted in development of first series of clinical inhibitors and tool compounds which continue to resolve and help understand the complex relationships between chromatin modification, control of gene expression and metabolic states.

Platt JL, Salama R, Smythies J, Choudhry H, Davies JO, Hughes JR, Ratcliffe PJ, Mole DR. 2016. Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters. EMBO Rep, 17 (10), pp. 1410-1421. | Citations: 16 (Web of Science Lite) | Show Abstract | Read more

Hypoxia-inducible factor (HIF) directs an extensive transcriptional cascade that transduces numerous adaptive responses to hypoxia. Pan-genomic analyses, using chromatin immunoprecipitation and transcript profiling, have revealed large numbers of HIF-binding sites that are generally associated with hypoxia-inducible transcripts, even over long chromosomal distances. However, these studies do not define the specific targets of HIF-binding sites and do not reveal how induction of HIF affects chromatin conformation over distantly connected functional elements. To address these questions, we deployed a recently developed chromosome conformation assay that enables simultaneous high-resolution analyses from multiple viewpoints. These assays defined specific long-range interactions between intergenic HIF-binding regions and one or more promoters of hypoxia-inducible genes, revealing the existence of multiple enhancer-promoter, promoter-enhancer, and enhancer-enhancer interactions. However, neither short-term activation of HIF by hypoxia, nor long-term stabilization of HIF in von Hippel-Lindau (VHL)-defective cells greatly alters these interactions, indicating that at least under these conditions, HIF can operate on preexisting patterns of chromatin-chromatin interactions that define potential transcriptional targets and permit rapid gene activation by hypoxic stress.

Gapp BV, Konopka T, Penz T, Dalal V, Bürckstümmer T, Bock C, Nijman SM. 2016. Parallel reverse genetic screening in mutant human cells using transcriptomics. Mol Syst Biol, 12 (8), pp. 879. | Citations: 4 (European Pubmed Central) | Show Abstract | Read more

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease-relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large-scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.

Miranda F, Mannion D, Liu S, Zheng Y, Mangala LS, Redondo C, Herrero-Gonzalez S, Xu R, Taylor C, Chedom DF et al. 2016. Salt-Inducible Kinase 2 Couples Ovarian Cancer Cell Metabolism with Survival at the Adipocyte-Rich Metastatic Niche. Cancer Cell, 30 (2), pp. 273-289. | Citations: 32 (Scopus) | Show Abstract | Read more

The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer.

Alexopoulou Z, Lang J, Perrett RM, Elschami M, Hurry MED, Kim HT, Mazaraki D, Szabo A, Kessler BM, Goldberg AL et al. 2016. Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease. Proc Natl Acad Sci U S A, 113 (32), pp. E4688-E4697. | Citations: 26 (Scopus) | Show Abstract | Read more

In Parkinson's disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. An important outstanding question is whether ubiquitination in Lewy bodies is directly relevant to α-synuclein trafficking or turnover and Parkinson's pathogenesis. By comparative analysis in human postmortem brains, we found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared with the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly colocalized with α-synuclein in endosomal membranes and, both in cells and after purification, it deubiquitinated K63-linked chains on α-synuclein. Knockdown of Usp8 in the Drosophila eye reduced α-synuclein levels and α-synuclein-induced eye toxicity. Accordingly, in human cells, Usp8 knockdown increased the lysosomal degradation of α-synuclein. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein-induced locomotor deficits and cell loss. These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism that reduces its lysosomal degradation in dopaminergic neurons and may contribute to α-synuclein accumulation in Lewy body disease.

Kaisar M, van Dullemen LFA, Thézénas M-L, Charles PD, Ploeg RJ, Kessler BM. 2016. Plasma Biomarker Profile Alterations during Variable Blood Storage. Clin Chem, 62 (9), pp. 1272-1274. | Citations: 1 (European Pubmed Central) | Read more

Chowdhury R, Leung IKH, Tian Y-M, Abboud MI, Ge W, Domene C, Cantrelle F-X, Landrieu I, Hardy AP, Pugh CW et al. 2016. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nat Commun, 7 (1), pp. 12673. | Citations: 26 (Scopus) | Show Abstract | Read more

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

Pinto-Fernandez A, Kessler BM. 2016. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets. Front Genet, 7 (JUL), pp. 133. | Citations: 22 (Scopus) | Show Abstract | Read more

Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

Lori L, Pasquo A, Lori C, Petrosino M, Chiaraluce R, Tallant C, Knapp S, Consalvi V. 2016. Effect of BET Missense Mutations on Bromodomain Function, Inhibitor Binding and Stability. PLoS One, 11 (7), pp. e0159180. | Citations: 6 (Web of Science Lite) | Show Abstract | Read more

Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of attention in the oncology field resulting in the rapid translation of early BET bromodomain inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism or found in cancer on BET bromodomain function and stability and the influence of these mutants on inhibitor binding. We found that most BET missense mutations localize to peripheral residues in the two terminal helices. Crystal structures showed that the three dimensional structure is not compromised by these mutations but mutations located in close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding. Most mutations affect significantly protein stability and tertiary structure in solution, suggesting new interactions and an alternative network of protein-protein interconnection as a consequence of single amino acid substitution. To our knowledge this is the first report studying the effect of mutations on bromodomain function and inhibitor binding.

Sdelci S, Lardeau C-H, Tallant C, Klepsch F, Klaiber B, Bennett J, Rathert P, Schuster M, Penz T, Fedorov O et al. 2016. Mapping the chemical chromatin reactivation landscape identifies BRD4-TAF1 cross-talk. Nat Chem Biol, 12 (7), pp. 504-510. | Citations: 14 (Web of Science Lite) | Show Abstract | Read more

Bromodomain-containing proteins of the BET family recognize histone lysine acetylation and mediate transcriptional activation of target genes such as the MYC oncogene. Pharmacological inhibitors of BET domains promise therapeutic benefits in a variety of cancers. We performed a high-diversity chemical compound screen for agents capable of modulating BRD4-dependent heterochromatization of a generic reporter in human cells. In addition to known and new compounds targeting BRD4, we identified small molecules that mimic BRD4 inhibition without direct engagement. One such compound was a potent inhibitor of the second bromodomain of TAF1. Using this inhibitor, we discovered that TAF1 synergizes with BRD4 to control proliferation of cancer cells, making TAF1 an attractive epigenetic target in cancers driven by MYC.

Montenegro RC, Clark PGK, Howarth A, Wan X, Ceroni A, Siejka P, Nunez-Alonso GA, Monteiro O, Rogers C, Gamble V et al. 2016. BET inhibition as a new strategy for the treatment of gastric cancer. Oncotarget, 7 (28), pp. 43997-44012. | Citations: 18 (Scopus) | Show Abstract | Read more

Gastric cancer is one of the most common malignancies and a leading cause of cancer death worldwide. The prognosis of stomach cancer is generally poor as this cancer is not very sensitive to commonly used chemotherapies. Epigenetic modifications play a key role in gastric cancer and contribute to the development and progression of this malignancy. In order to explore new treatment options in this target area we have screened a library of epigenetic inhibitors against gastric cancer cell lines and identified inhibitors for the BET family of bromodomains as potent inhibitors of gastric cancer cell proliferations. Here we show that both the pan-BET inhibitor (+)-JQ1 as well as a newly developed specific isoxazole inhibitor, PNZ5, showed potent inhibition of gastric cancer cell growth. Intriguingly, we found differences in the antiproliferative response between gastric cancer cells tested derived from Brazilian patients as compared to those from Asian patients, the latter being largely resistant to BET inhibition. As BET inhibitors are entering clinical trials these findings provide the first starting point for future therapies targeting gastric cancer.

Chang AN, Mahajan P, Knapp S, Barton H, Sweeney HL, Kamm KE, Stull JT. 2016. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities. Proc Natl Acad Sci U S A, 113 (27), pp. E3824-E3833. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.

Vukovic S, Brennan PE, Huggins DJ. 2016. Exploring the role of water in molecular recognition: predicting protein ligandability using a combinatorial search of surface hydration sites. J Phys Condens Matter, 28 (34), pp. 344007. | Citations: 13 (Scopus) | Show Abstract | Read more

The interaction between any two biological molecules must compete with their interaction with water molecules. This makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. A small molecule binding to a protein is able to recognize a unique binding site on a protein by displacing bound water molecules from specific hydration sites. Quantifying the interactions of these water molecules allows us to estimate the potential of the protein to bind a small molecule. This is referred to as ligandability. In the study, we describe a method to predict ligandability by performing a search of all possible combinations of hydration sites on protein surfaces. We predict ligandability as the summed binding free energy for each of the constituent hydration sites, computed using inhomogeneous fluid solvation theory. We compared the predicted ligandability with the maximum observed binding affinity for 20 proteins in the human bromodomain family. Based on this comparison, it was determined that effective inhibitors have been developed for the majority of bromodomains, in the range from 10 to 100 nM. However, we predict that more potent inhibitors can be developed for the bromodomains BPTF and BRD7 with relative ease, but that further efforts to develop inhibitors for ATAD2 will be extremely challenging. We have also made predictions for the 14 bromodomains with no reported small molecule K d values by isothermal titration calorimetry. The calculations predict that PBRM1(1) will be a challenging target, while others such as TAF1L(2), PBRM1(4) and TAF1(2), should be highly ligandable. As an outcome of this work, we assembled a database of experimental maximal K d that can serve as a community resource assisting medicinal chemistry efforts focused on BRDs. Effective prediction of ligandability would be a very useful tool in the drug discovery process.

Faherty N, Benson M, Sharma E, Lee A, Howarth A, Lockstone H, Ebner D, Bhattacharya S. 2016. Negative autoregulation of BMP dependent transcription by SIN3B splicing reveals a role for RBM39. Sci Rep, 6 (1), pp. 28210. | Show Abstract | Read more

BMP signalling is negatively autoregulated by several genes including SMAD6, Noggin and Gremlin, and autoregulators are possible targets for enhancing BMP signalling in disorders such as fibrosis and pulmonary hypertension. To identify novel negative regulators of BMP signalling, we used siRNA screening in mouse C2C12 cells with a BMP-responsive luciferase reporter. Knockdown of several splicing factors increased BMP4-dependent transcription and target gene expression. Knockdown of RBM39 produced the greatest enhancement in BMP activity. Transcriptome-wide RNA sequencing identified a change in Sin3b exon usage after RBM39 knockdown. SIN3B targets histone deacetylases to chromatin to repress transcription. In mouse, Sin3b produces long and short isoforms, with the short isoform lacking the ability to recruit HDACs. BMP4 induced a shift in SIN3B expression to the long isoform, and this change in isoform ratio was prevented by RBM39 knockdown. Knockdown of long isoform SIN3B enhanced BMP4-dependent transcription, whereas knockdown of the short isoform did not. We propose that BMP4-dependent transcription is negatively autoregulated in part by SIN3B alternative splicing, and that RBM39 plays a role in this process.

Grijzenhout A, Godwin J, Koseki H, Gdula MR, Szumska D, McGouran JF, Bhattacharya S, Kessler BM, Brockdorff N, Cooper S. 2016. Functional analysis of AEBP2, a PRC2 Polycomb protein, reveals a Trithorax phenotype in embryonic development and in ESCs. Development, 143 (15), pp. 2716-2723. | Citations: 20 (Scopus) | Show Abstract | Read more

The Polycomb repressive complexes PRC1 and PRC2 are key mediators of heritable gene silencing in multicellular organisms. Here, we characterise AEBP2, a known PRC2 co-factor which, in vitro, has been shown to stimulate PRC2 activity. We show that AEBP2 localises specifically to PRC2 target loci, including the inactive X chromosome. Proteomic analysis confirms that AEBP2 associates exclusively with PRC2 complexes. However, analysis of embryos homozygous for a targeted mutation of Aebp2 unexpectedly revealed a Trithorax phenotype, normally linked to antagonism of Polycomb function. Consistent with this, we observe elevated levels of PRC2-mediated histone H3K27 methylation at target loci in Aebp2 mutant embryonic stem cells (ESCs). We further demonstrate that mutant ESCs assemble atypical hybrid PRC2 subcomplexes, potentially accounting for enhancement of Polycomb activity, and suggesting that AEBP2 normally plays a role in defining the mutually exclusive composition of PRC2 subcomplexes.

Mathea S, Abdul Azeez KR, Salah E, Tallant C, Wolfreys F, Konietzny R, Fischer R, Lou HJ, Brennan PE, Schnapp G et al. 2016. Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib. ACS Chem Biol, 11 (6), pp. 1595-1602. | Citations: 6 (Scopus) | Show Abstract | Read more

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here, we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The cocrystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions -1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs.

Raducu M, Fung E, Serres S, Infante P, Barberis A, Fischer R, Bristow C, Thézénas M-L, Finta C, Christianson JC et al. 2016. SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development. EMBO J, 35 (13), pp. 1400-1416. | Citations: 12 (European Pubmed Central) | Show Abstract | Read more

Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.

Milhas S, Raux B, Betzi S, Derviaux C, Roche P, Restouin A, Basse M-J, Rebuffet E, Lugari A, Badol M et al. 2016. Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery. ACS Chem Biol, 11 (8), pp. 2140-2148. | Citations: 6 (Web of Science Lite) | Show Abstract | Read more

Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.

Johansson C, Velupillai S, Tumber A, Szykowska A, Hookway ES, Nowak RP, Strain-Damerell C, Gileadi C, Philpott M, Burgess-Brown N et al. 2016. Structural analysis of human KDM5B guides histone demethylase inhibitor development. Nat Chem Biol, 12 (7), pp. 539-545. | Citations: 40 (Web of Science Lite) | Show Abstract | Read more

Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe(2+)-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25-100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.

Frise MC, Cheng H-Y, Nickol AH, Curtis MK, Pollard KA, Roberts DJ, Ratcliffe PJ, Dorrington KL, Robbins PA. 2016. Clinical iron deficiency disturbs normal human responses to hypoxia. J Clin Invest, 126 (6), pp. 2139-2150. | Citations: 27 (Web of Science Lite) | Show Abstract | Read more

BACKGROUND: Iron bioavailability has been identified as a factor that influences cellular hypoxia sensing, putatively via an action on the hypoxia-inducible factor (HIF) pathway. We therefore hypothesized that clinical iron deficiency would disturb integrated human responses to hypoxia. METHODS: We performed a prospective, controlled, observational study of the effects of iron status on hypoxic pulmonary hypertension. Individuals with absolute iron deficiency (ID) and an iron-replete (IR) control group were exposed to two 6-hour periods of isocapnic hypoxia. The second hypoxic exposure was preceded by i.v. infusion of iron. Pulmonary artery systolic pressure (PASP) was serially assessed with Doppler echocardiography. RESULTS: Thirteen ID individuals completed the study and were age- and sex-matched with controls. PASP did not differ by group or study day before each hypoxic exposure. During the first 6-hour hypoxic exposure, the rise in PASP was 6.2 mmHg greater in the ID group (absolute rises 16.1 and 10.7 mmHg, respectively; 95% CI for difference, 2.7-9.7 mmHg, P = 0.001). Intravenous iron attenuated the PASP rise in both groups; however, the effect was greater in ID participants than in controls (absolute reductions 11.1 and 6.8 mmHg, respectively; 95% CI for difference in change, -8.3 to -0.3 mmHg, P = 0.035). Serum erythropoietin responses to hypoxia also differed between groups. CONCLUSION: Clinical iron deficiency disturbs normal responses to hypoxia, as evidenced by exaggerated hypoxic pulmonary hypertension that is reversed by subsequent iron administration. Disturbed hypoxia sensing and signaling provides a mechanism through which iron deficiency may be detrimental to human health. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01847352). FUNDING: M.C. Frise is the recipient of a British Heart Foundation Clinical Research Training Fellowship (FS/14/48/30828). K.L. Dorrington is supported by the Dunhill Medical Trust (R178/1110). D.J. Roberts was supported by R&D funding from National Health Service (NHS) Blood and Transplant and a National Institute for Health Research (NIHR) Programme grant (RP-PG-0310-1004). This research was funded by the NIHR Oxford Biomedical Research Centre Programme.

Frago S, Nicholls RD, Strickland M, Hughes J, Williams C, Garner L, Surakhy M, Maclean R, Rezgui D, Prince SN et al. 2016. Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist. Proc Natl Acad Sci U S A, 113 (20), pp. E2766-E2775. | Citations: 8 (Web of Science Lite) | Show Abstract | Read more

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.

Akhtar MZ, Huang H, Kaisar M, Lo Faro ML, Rebolledo R, Morten K, Heather LC, Dona A, Leuvenink HG, Fuggle SV et al. 2016. Using an Integrated -Omics Approach to Identify Key Cellular Processes That Are Disturbed in the Kidney After Brain Death. Am J Transplant, 16 (5), pp. 1421-1440. | Citations: 3 (European Pubmed Central) | Show Abstract | Read more

In an era where we are becoming more reliant on vulnerable kidneys for transplantation from older donors, there is an urgent need to understand how brain death leads to kidney dysfunction and, hence, how this can be prevented. Using a rodent model of hemorrhagic stroke and next-generation proteomic and metabolomic technologies, we aimed to delineate which key cellular processes are perturbed in the kidney after brain death. Pathway analysis of the proteomic signature of kidneys from brain-dead donors revealed large-scale changes in mitochondrial proteins that were associated with altered mitochondrial activity and morphological evidence of mitochondrial injury. We identified an increase in a number of glycolytic proteins and lactate production, suggesting a shift toward anaerobic metabolism. Higher amounts of succinate were found in the brain death group, in conjunction with increased markers of oxidative stress. We characterized the responsiveness of hypoxia inducible factors and found this correlated with post-brain death mean arterial pressures. Brain death leads to metabolic disturbances in the kidney and alterations in mitochondrial function and reactive oxygen species generation. This metabolic disturbance and alteration in mitochondrial function may lead to further cellular injury. Conditioning the brain-dead organ donor by altering metabolism could be a novel approach to ameliorate this brain death-induced kidney injury.

Lavogina D, Kestav K, Chaikuad A, Heroven C, Knapp S, Uri A. 2016. Co-crystal structures of the protein kinase haspin with bisubstrate inhibitors. Acta Crystallogr F Struct Biol Commun, 72 (Pt 5), pp. 339-345. | Citations: 3 (Web of Science Lite) | Show Abstract | Read more

Haspin is a mitotic protein kinase that is responsible for the phosphorylation of Thr3 of histone H3, thereby creating a recognition motif for docking of the chromosomal passenger complex that is crucial for the progression of cell division. Here, two high-resolution models of haspin with previously reported inhibitors consisting of an ATP analogue and a histone H3(1-7) peptide analogue are presented. The structures of the complexes confirm the bisubstrate character of the inhibitors by revealing the signature binding modes of the moieties targeting the ATP-binding site and the protein substrate-binding site of the kinase. This is the first structural model of a bisubstrate inhibitor targeting haspin. The presented structural data represent a model for the future development of more specific haspin inhibitors.

Sutherell CL, Tallant C, Monteiro OP, Yapp C, Fuchs JE, Fedorov O, Siejka P, Müller S, Knapp S, Brenton JD et al. 2016. Identification and Development of 2,3-Dihydropyrrolo[1,2-a]quinazolin-5(1H)-one Inhibitors Targeting Bromodomains within the Switch/Sucrose Nonfermenting Complex. J Med Chem, 59 (10), pp. 5095-5101. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

Bromodomain containing proteins PB1, SMARCA4, and SMARCA2 are important components of SWI/SNF chromatin remodeling complexes. We identified bromodomain inhibitors that target these proteins and display unusual binding modes involving water displacement from the KAc binding site. The best compound binds the fifth bromodomain of PB1 with a KD of 124 nM, SMARCA2B and SMARCA4 with KD values of 262 and 417 nM, respectively, and displays excellent selectivity over bromodomains other than PB1, SMARCA2, and SMARCA4.

Gerstenberger BS, Trzupek JD, Tallant C, Fedorov O, Filippakopoulos P, Brennan PE, Fedele V, Martin S, Picaud S, Rogers C et al. 2016. Identification of a Chemical Probe for Family VIII Bromodomains through Optimization of a Fragment Hit. J Med Chem, 59 (10), pp. 4800-4811. | Citations: 21 (Scopus) | Show Abstract | Read more

The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.

Cole MA, Abd Jamil AH, Heather LC, Murray AJ, Sutton ER, Slingo M, Sebag-Montefiore L, Tan SC, Aksentijević D, Gildea OS et al. 2016. On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury. FASEB J, 30 (8), pp. 2684-2697. | Citations: 8 (European Pubmed Central) | Show Abstract | Read more

The role of peroxisome proliferator-activated receptor α (PPARα)-mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 wk before in vivo contractile function was measured using cine MRI. In isolated, perfused hearts, energetics were measured using (31)P magnetic resonance spectroscopy (MRS), and glycolysis and fatty acid oxidation were measured using [(3)H] labeling. Compared with a normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation, and mitochondrial uncoupling protein 3 (UCP3) levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations ([ATP]) and thereby, ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation, and UCP3 levels with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high-fat diet, with the result that [ATP] and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have occurred already in PPARα-deficient (PPARα(-/-)) mouse hearts and sustained function in hypoxia despite an inability for further metabolic remodeling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodeling in hypoxia, but is prevented by dietary fat.-Cole, M. A., Abd Jamil, A. H., Heather, L. C., Murray, A. J., Sutton, E. R., Slingo, M., Sebag-Montefiore, L., Tan, S. C., Aksentijević, D., Gildea, O. S., Stuckey, D. J., Yeoh, K. K., Carr, C. A., Evans, R. D., Aasum, E., Schofield, C. J., Ratcliffe, P. J., Neubauer, S., Robbins, P. A., Clarke, K. On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.

Lochmatter C, Fischer R, Charles PD, Yu Z, Powrie F, Kessler BM. 2016. Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes. Sci Rep, 6 (1), pp. 24491. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn's disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation.

Pan J, Bishop T, Ratcliffe PJ, Yeger H, Cutz E. 2016. Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice. Hypoxia (Auckl), 4 pp. 69-80. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)-hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1-/- mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1-/-, Phd2+/-, and Phd3-/- mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1-/- and Phd3-/- mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/- mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.

Chung VY, Konietzny R, Charles P, Kessler B, Fischer R, Turney BW. 2016. Proteomic changes in response to crystal formation in Drosophila Malpighian tubules. Fly (Austin), 10 (2), pp. 91-100. | Citations: 1 (Web of Science Lite) | Show Abstract | Read more

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.

Vukovic M, Sepulveda C, Subramani C, Guitart AV, Mohr J, Allen L, Panagopoulou TI, Paris J, Lawson H, Villacreces A et al. 2016. Adult hematopoietic stem cells lacking Hif-1α self-renew normally. Blood, 127 (23), pp. 2841-2846. | Citations: 13 (European Pubmed Central) | Show Abstract | Read more

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance.

Esvan YJ, Zeinyeh W, Boibessot T, Nauton L, Théry V, Knapp S, Chaikuad A, Loaëc N, Meijer L, Anizon F et al. 2016. Discovery of pyrido[3,4-g]quinazoline derivatives as CMGC family protein kinase inhibitors: Design, synthesis, inhibitory potency and X-ray co-crystal structure. Eur J Med Chem, 118 pp. 170-177. | Citations: 10 (Scopus) | Show Abstract | Read more

The design and synthesis of new pyrido[3,4-g]quinazoline derivatives is described as well as their protein kinase inhibitory potencies toward five CMGC family members (CDK5, CK1, GSK3, CLK1 and DYRK1A). The interest for this original tricyclic heteroaromatic scaffold as modulators of CLK1/DYRK1A activity was validated by nanomolar potencies (compounds 12 and 13). CLK1 co-crystal structures with two inhibitors revealed the binding mode of these compounds within the ATP-binding pocket.

Schödel J, Grampp S, Maher ER, Moch H, Ratcliffe PJ, Russo P, Mole DR. 2016. Hypoxia, Hypoxia-inducible Transcription Factors, and Renal Cancer. Eur Urol, 69 (4), pp. 646-657. | Citations: 57 (Web of Science Lite) | Show Abstract | Read more

CONTEXT: Renal cancer is a common urologic malignancy, and therapeutic options for metastatic disease are limited. Most clear cell renal cell carcinomas (ccRCC) are associated with loss of von Hippel-Lindau tumor suppressor (pVHL) function and deregulation of hypoxia pathways. OBJECTIVE: This review summarizes recent evidence from genetic and biological studies showing that hypoxia and hypoxia-related pathways play critical roles in the development and progress of renal cancer. EVIDENCE ACQUISITION: We used a systematic search for articles using the keywords hypoxia, HIF, renal cancer, and VHL. EVIDENCE SYNTHESIS: Identification of the tumor suppressor pVHL has allowed the characterization of important ccRCC-associated pathways. pVHL targets α-subunits of hypoxia-inducible transcription factors (HIF) for proteasomal degradation. The two main HIF-α isoforms have opposing effects on RCC biology, possibly through distinct interactions with additional oncogenes. Furthermore, HIF-1α activity is commonly diminished by chromosomal deletion in ccRCCs, and increased HIF-1 activity reduces tumor burden in xenograft tumor models. Conversely, polymorphisms at the HIF-2α gene locus predispose to the development of ccRCCs, and HIF-2α promotes tumor growth. Genetic studies have revealed a prominent role for chromatin-modifying enzyme genes in ccRCC, and these may further modulate specific aspects of the HIF response. This suggests that, rather than global activation of HIF, specific components of the response are important in promoting kidney cancer. Some of these processes are already targets for current therapeutic strategies, and further dissection of this pathway might yield novel methods of treating RCC. CONCLUSIONS: In contrast to many tumor types, HIF-1α and HIF-2α have opposing effects in ccRCC biology, with HIF-1α acting as a tumor suppressor and HIF-2α acting as an oncogene. The overall effect of VHL inactivation will depend on fine-tuning of the HIF response. PATIENT SUMMARY: High levels of hypoxia-inducible transcription factors (HIF) are particularly important in the clear cell type of kidney cancer, in which they are no longer properly regulated by the von Hippel-Lindau protein. The two HIF-α proteins have opposing effects on tumor evolution.

Van Dullemen L, Kaisar M, Akhtar Z, Lo Faro L, Huang H, Leuvenink H, Kessler B, Ploeg R. 2016. ACTIVATION OF CYTOPROTECTION MECHANISMS IN DBD DONOR KIDNEYS DEFINE KIDNEY FUNCTION IN TRANSPLANT RECIPIENTS- A STUDY USING CLINICAL SAMPLES OBTAINED FROM THE QUOD BIOBANK TRANSPLANT INTERNATIONAL, 29 pp. 3-3.

Coutandin D, Osterburg C, Srivastav RK, Sumyk M, Kehrloesser S, Gebel J, Tuppi M, Hannewald J, Schäfer B, Salah E et al. 2016. Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level. Elife, 5 (MARCH2016), | Citations: 17 (Web of Science Lite) | Show Abstract | Read more

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.

Martin LJ, Koegl M, Bader G, Cockcroft X-L, Fedorov O, Fiegen D, Gerstberger T, Hofmann MH, Hohmann AF, Kessler D et al. 2016. Structure-Based Design of an in Vivo Active Selective BRD9 Inhibitor. J Med Chem, 59 (10), pp. 4462-4475. | Citations: 56 (Scopus) | Show Abstract | Read more

Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

Hodson EJ, Nicholls LG, Turner PJ, Llyr R, Fielding JW, Douglas G, Ratnayaka I, Robbins PA, Pugh CW, Buckler KJ et al. 2016. Regulation of ventilatory sensitivity and carotid body proliferation in hypoxia by the PHD2/HIF-2 pathway. J Physiol, 594 (5), pp. 1179-1195. | Citations: 18 (European Pubmed Central) | Show Abstract | Read more

Ventilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia-inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF-1α and HIF-2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen-treated Phd2(f/f);Rosa26(+/CreERT2) mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min(-1) g(-1) in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF-2α, but not HIF-1α. Furthermore, inducible inactivation of HIF-2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min(-1) g(-1) in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine-positive cells mm(-2) in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF-2α enzyme-substrate couple in modulating ventilatory sensitivity to hypoxia.

Cox OB, Krojer T, Collins P, Monteiro O, Talon R, Bradley A, Fedorov O, Amin J, Marsden BD, Spencer J et al. 2016. A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain. Chem Sci, 7 (3), pp. 2322-2330. | Citations: 23 (Scopus) | Show Abstract | Read more

Research into the chemical biology of bromodomains has been driven by the development of acetyl-lysine mimetics. The ligands are typically anchored by binding to a highly conserved asparagine residue. Atypical bromodomains, for which the asparagine is mutated, have thus far proven elusive targets, including PHIP(2) whose parent protein, PHIP, has been linked to disease progression in diabetes and cancers. The PHIP(2) binding site contains a threonine in place of asparagine, and solution screening have yielded no convincing hits. We have overcome this hurdle by combining the sensitivity of X-ray crystallography, used as the primary fragment screen, with a strategy for rapid follow-up synthesis using a chemically-poised fragment library, which allows hits to be readily modified by parallel chemistry both peripherally and in the core. Our approach yielded the first reported hit compounds of PHIP(2) with measurable IC50 values by an AlphaScreen competition assay. The follow-up libraries of four poised fragment hits improved potency into the sub-mM range while showing good ligand efficiency and detailed structural data.

Clark PGK, Dixon DJ, Brennan PE. 2016. Development of chemical probes for the bromodomains of BRD7 and BRD9. Drug Discov Today Technol, 19 pp. 73-80. | Citations: 5 (Scopus) | Show Abstract | Read more

The bromodomain family of proteins are 'readers' of acetylated lysines of histones, a key mark in the epigenetic code of gene regulation. Without high quality chemical probes with which to study these proteins, their biological function, and potential use in therapeutics, remains unknown. Recently, a number of chemical ligands were reported for the previously unprobed bromodomain proteins BRD7 and BRD9. Herein the development and characterisation of probes against these proteins is detailed, including the preliminary biological activity of BRD7 and BRD9 assessed using these probes. Future studies utilising these chemically-diverse compounds in parallel will allow for a confident assessment of the role of BRD7/9, and give multiple entry points into any subsequent pharmaceutical programs.

Wildenberg M, Levin A, Ceroni A, Guo Z, Bloemendaal F, Ebner D, van den Brink G. 2016. Large-scale drug screen reveals benzimidazole anti-helminthics as potential anti-TNF co-therapy JOURNAL OF CROHNS & COLITIS, 10 pp. S41-S42.

Rahighi S, Braunstein I, Ternette N, Kessler B, Kawasaki M, Kato R, Matsui T, Weiss TM, Stanhill A, Wakatsuki S. 2016. Selective Binding of AIRAPL Tandem UIMs to Lys48-Linked Tri-Ubiquitin Chains. Structure, 24 (3), pp. 412-422. | Citations: 7 (Web of Science Lite) | Show Abstract | Read more

Lys48-linked ubiquitin chains act as the main targeting signals for protein degradation by the proteasome. Here we report selective binding of AIRAPL, a protein that associates with the proteasome upon exposure to arsenite, to Lys48-linked tri-ubiquitin chains. AIRAPL comprises two ubiquitin-interacting motifs in tandem (tUIMs) that are linked through a flexible inter-UIM region. In the complex crystal structure UIM1 binds the proximal ubiquitin, whereas UIM2 (the double-sided UIM) binds non-symmetrically to the middle and distal ubiquitin moieties on either side of the helix. Specificity of AIRAPL for Lys48-linked ubiquitin chains is determined by UIM2, and the flexible inter-UIM linker increases avidity by placing the two UIMs in an orientation that facilitates binding of the third ubiquitin to UIM1. Unlike middle and proximal ubiquitins, distal ubiquitin binds UIM2 through a novel surface, which leaves the Ile44 hydrophobic patch accessible for binding to the proteasomal ubiquitin receptors.

Ghari F, Quirke A-M, Munro S, Kawalkowska J, Picaud S, McGouran J, Subramanian V, Muth A, Williams R, Kessler B et al. 2016. Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response. Sci Adv, 2 (2), pp. e1501257. | Citations: 26 (Scopus) | Show Abstract | Read more

Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.

McAllister TE, England KS, Hopkinson RJ, Brennan PE, Kawamura A, Schofield CJ. 2016. Recent Progress in Histone Demethylase Inhibitors. J Med Chem, 59 (4), pp. 1308-1329. | Citations: 76 (Scopus) | Show Abstract | Read more

There is increasing interest in targeting histone N-methyl-lysine demethylases (KDMs) with small molecules both for the generation of probes for target exploration and for therapeutic purposes. Here we update on previous reviews on the inhibition of the lysine-specific demethylases (LSDs or KDM1s) and JmjC families of N-methyl-lysine demethylases (JmjC KDMs, KDM2-7), focusing on the academic and patent literature from 2014 to date. We also highlight recent biochemical, biological, and structural studies which are relevant to KDM inhibitor development.

Chan MC, Holt-Martyn JP, Schofield CJ, Ratcliffe PJ. 2016. Pharmacological targeting of the HIF hydroxylases--A new field in medicine development. Mol Aspects Med, 47-48 pp. 54-75. | Citations: 47 (Web of Science Lite) | Show Abstract | Read more

In human cells oxygen levels are 'sensed' by a set of ferrous iron and 2-oxoglutarate dependent dioxygenases. These enzymes regulate a broad range of cellular and systemic responses to hypoxia by catalysing the post-translational hydroxylation of specific residues in the alpha subunits of hypoxia inducible factor (HIF) transcriptional complexes. The HIF hydroxylases are now the subject of pharmaceutical targeting by small molecule inhibitors that aim to activate or augment the endogenous HIF transcriptional response for the treatment of anaemia and other hypoxic human diseases. Here we consider the rationale for this therapeutic strategy from the biochemical, biological and medical perspectives. We outline structural and mechanistic considerations that are relevant to the design of HIF hydroxylase inhibitors, including likely determinants of specificity, and review published reports on their activity in pre-clinical models and clinical trials.

Sorrell FJ, Szklarz M, Abdul Azeez KR, Elkins JM, Knapp S. 2016. Family-wide Structural Analysis of Human Numb-Associated Protein Kinases. Structure, 24 (3), pp. 401-411. | Citations: 8 (Web of Science Lite) | Show Abstract | Read more

The highly diverse Numb-associated kinase (NAK) family has been linked to broad cellular functions including receptor-mediated endocytosis, Notch pathway modulation, osteoblast differentiation, and dendrite morphogenesis. Consequently, NAK kinases play a key role in a diverse range of diseases from Parkinson's and prostate cancer to HIV. Due to the plasticity of this kinase family, NAK kinases are often inhibited by approved or investigational drugs and have been associated with side effects, but they are also potential drug targets. The presence of cysteine residues in some NAK family members provides the possibility for selective targeting via covalent inhibition. Here we report the first high-resolution structures of kinases AAK1 and BIKE in complex with two drug candidates. The presented data allow a comprehensive structural characterization of the NAK kinase family and provide the basis for rational design of selective NAK inhibitors.

Brennan PE. 2016. Deciphering the true antiproliferative target of an MK2 activation inhibitor in glioblastoma. Cell Death Dis, 7 (1), pp. e2069. | Citations: 2 (Web of Science Lite) | Read more

Kumar R, Li D-Q, Müller S, Knapp S. 2016. Epigenomic regulation of oncogenesis by chromatin remodeling. Oncogene, 35 (34), pp. 4423-4436. | Citations: 32 (Scopus) | Show Abstract | Read more

Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy.

Zuercher WJ, Elkins JM, Knapp S. 2016. The Intersection of Structural and Chemical Biology - An Essential Synergy. Cell Chem Biol, 23 (1), pp. 173-182. | Citations: 7 (Web of Science Lite) | Show Abstract | Read more

The continual improvement in our ability to generate high resolution structural models of biological molecules has stimulated and supported innovative chemical biology projects that target increasingly challenging ligand interaction sites. In this review we outline some of the recent developments in chemical biology and rational ligand design and show selected examples that illustrate the synergy between these research areas.

Aldeghi M, Heifetz A, Bodkin MJ, Knapp S, Biggin PC. 2016. Accurate calculation of the absolute free energy of binding for drug molecules. Chem Sci, 7 (1), pp. 207-218. | Citations: 63 (Scopus) | Show Abstract | Read more

Accurate prediction of binding affinities has been a central goal of computational chemistry for decades, yet remains elusive. Despite good progress, the required accuracy for use in a drug-discovery context has not been consistently achieved for drug-like molecules. Here, we perform absolute free energy calculations based on a thermodynamic cycle for a set of diverse inhibitors binding to bromodomain-containing protein 4 (BRD4) and demonstrate that a mean absolute error of 0.6 kcal mol-1 can be achieved. We also show a similar level of accuracy (1.0 kcal mol-1) can be achieved in pseudo prospective approach. Bromodomains are epigenetic mark readers that recognize acetylation motifs and regulate gene transcription, and are currently being investigated as therapeutic targets for cancer and inflammation. The unprecedented accuracy offers the exciting prospect that the binding free energy of drug-like compounds can be predicted for pharmacologically relevant targets.

Chaikuad A, Lang S, Brennan PE, Temperini C, Fedorov O, Hollander J, Nachane R, Abell C, Müller S, Siegal G, Knapp S. 2016. Structure-Based Identification of Inhibitory Fragments Targeting the p300/CBP-Associated Factor Bromodomain. J Med Chem, 59 (4), pp. 1648-1653. | Citations: 17 (Scopus) | Show Abstract | Read more

The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.

Bavetsias V, Lanigan RM, Ruda GF, Atrash B, McLaughlin MG, Tumber A, Mok NY, Le Bihan Y-V, Dempster S, Boxall KJ et al. 2016. 8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors. J Med Chem, 59 (4), pp. 1388-1409. | Citations: 36 (Scopus) | Show Abstract | Read more

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

Willkomm J, Orchard KL, Reynal A, Pastor E, Durrant JR, Reisner E. 2016. Dye-sensitised semiconductors modified with molecular catalysts for light-driven H2 production. Chem Soc Rev, 45 (1), pp. 9-23. | Citations: 37 (European Pubmed Central) | Show Abstract | Read more

The development of synthetic systems for the conversion of solar energy into chemical fuels is a research goal that continues to attract growing interest owing to its potential to provide renewable and storable energy in the form of a 'solar fuel'. Dye-sensitised photocatalysis (DSP) with molecular catalysts is a relatively new approach to convert sunlight into a fuel such as H2 and is based on the self-assembly of a molecular dye and electrocatalyst on a semiconductor nanoparticle. DSP systems combine advantages of both homogenous and heterogeneous photocatalysis, with the molecular components providing an excellent platform for tuning activity and understanding performance at defined catalytic sites, whereas the semiconductor bridge ensures favourable multi-electron transfer kinetics between the dye and the fuel-forming electrocatalyst. In this tutorial review, strategies and challenges for the assembly of functional molecular DSP systems and experimental techniques for their evaluation are explained. Current understanding of the factors governing electron transfer across inorganic-molecular interfaces is described and future directions and challenges for this field are outlined.

Raux B, Voitovich Y, Derviaux C, Lugari A, Rebuffet E, Milhas S, Priet S, Roux T, Trinquet E, Guillemot J-C et al. 2016. Exploring Selective Inhibition of the First Bromodomain of the Human Bromodomain and Extra-terminal Domain (BET) Proteins. J Med Chem, 59 (4), pp. 1634-1641. | Citations: 30 (Scopus) | Show Abstract | Read more

A midthroughput screening follow-up program targeting the first bromodomain of the human BRD4 protein, BRD4(BD1), identified an acetylated-mimic xanthine derivative inhibitor. This compound binds with an affinity in the low micromolar range yet exerts suitable unexpected selectivity in vitro against the other members of the bromodomain and extra-terminal domain (BET) family. A structure-based program pinpointed a role of the ZA loop, paving the way for the development of potent and selective BET-BRDi probes.

Huber KVM, Superti-Furga G. 2016. Profiling of Small Molecules by Chemical Proteomics. Methods Mol Biol, 1394 pp. 211-218. | Citations: 3 (European Pubmed Central) | Show Abstract | Read more

Chemical proteomics provides a powerful means to gain systems-level insight into the mode of action of small molecules and/or natural products. In contrast to high-throughput screening efforts which only interrogate selected subproteomes such as kinases and often only consider individual domains, the methodology presented herein allows for the determination of the molecular targets of small molecules or drugs in a more relevant physiological setting. As such, the compound of interest is exposed to the entire variety of cellular proteins considering all naturally occurring posttranslational modifications and activation states. Samples prepared according to the procedures described in this protocol are compatible with lysates from cultured cell lines, primary cells, or samples from biopsies. In combination with state-of-the-art mass spectrometry techniques this approach grants access to a comprehensive view of small molecule-target protein interactions.

Petersen LK, Blakskjaer P, Chaikuad A, Christensen AB, Dietvorst J, Holmkvist J, Knapp S, Korinek M, Larsen LK, Pedersen AE et al. 2016. Novel p38 alpha MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library MEDCHEMCOMM, 7 (7), pp. 1332-1339. | Citations: 13 (Scopus) | Show Abstract | Read more

<p>A DNA-encoded small-molecule library was prepared using yoctoReactor technology followed by binder trap enrichment to identify selective inhibitors with nanomolar potencies against p38α MAP kinase.</p>

Ceroni A, Higgins GS, Ebner DV. 2016. In Vitro-Pooled shRNA Screening to Identify Determinants of Radiosensitivity. Methods Mol Biol, 1470 pp. 103-119. | Show Abstract | Read more

Short hairpin RNA (shRNA)-pooled screening is a valuable and cost-effective tool for assaying the contribution of individual genes to cell viability and proliferation on a genomic scale. Here we describe the key considerations for the design and execution of a pooled shRNA screen to identify determinants of radiosensitivity.

Keating SM, Heitman JW, Wu S, Deng X, Stacey AR, Zahn RC, de la Rosa M, Finstad SL, Lifson JD, Piatak M et al. 2016. Magnitude and quality of cytokine and chemokine storm during acute infection distinguish nonprogressive and progressive simian immunodeficiency virus infections of nonhuman primates Journal of Virology, 90 (22), pp. 10339-10350. | Citations: 2 (Scopus) | Show Abstract | Read more

© 2016, American Society for Microbiology. All Rights Reserved. Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δnef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony- stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP- 1β), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δnef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression.

Schwenzer A, Jiang X, Mikuls TR, Payne JB, Sayles HR, Quirke A-M, Kessler BM, Fischer R, Venables PJ, Lundberg K, Midwood KS. 2016. Identification of an immunodominant peptide from citrullinated tenascin-C as a major target for autoantibodies in rheumatoid arthritis. Ann Rheum Dis, 75 (10), pp. 1876-1883. | Citations: 16 (Web of Science Lite) | Show Abstract | Read more

OBJECTIVES: We investigated whether citrullinated tenascin-C (cTNC), an extracellular matrix protein expressed at high levels in the joints of patients with rheumatoid arthritis (RA), is a target for the autoantibodies in RA. METHODS: Citrullinated sites were mapped by mass spectrometry in the fibrinogen-like globe (FBG) domain of tenascin-C treated with peptidylarginine deiminases (PAD) 2 and 4. Antibodies to cyclic peptides containing citrullinated sites were screened in sera from patients with RA by ELISA. Potential cross-reactivity with well-established anticitrullinated protein antibody (ACPA) epitopes was tested by inhibition assays. The autoantibody response to one immunodominant cTNC peptide was then analysed in 101 pre-RA sera (median 7 years before onset) and two large independent RA cohorts. RESULTS: Nine arginine residues within FBG were citrullinated by PAD2 and PAD4. Two immunodominant peptides cTNC1 (VFLRRKNG-cit-ENFYQNW) and cTNC5 (EHSIQFAEMKL-cit-PSNF-cit-NLEG-cit-cit-KR) were identified. Antibodies to both showed limited cross-reactivity with ACPA epitopes from α-enolase, vimentin and fibrinogen, and no reactivity with citrullinated fibrinogen peptides sharing sequence homology with FBG. cTNC5 antibodies were detected in 18% of pre-RA sera, and in 47% of 1985 Swedish patients with RA and 51% of 287 North American patients with RA. The specificity was 98% compared with 160 healthy controls and 330 patients with osteoarthritis. CONCLUSIONS: There are multiple citrullination sites in the FBG domain of tenascin-C. Among these, one epitope is recognised by autoantibodies that are detected years before disease onset, and which may serve as a useful biomarker to identify ACPA-positive patients with high sensitivity and specificity in established disease.

Ying S, Chen Z, Medhurst AL, Neal JA, Bao Z, Mortusewicz O, McGouran J, Song X, Shen H, Hamdy FC et al. 2016. DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair. Cancer Res, 76 (5), pp. 1078-1088. | Citations: 29 (Scopus) | Show Abstract | Read more

A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved.

Knapp S. 2016. Emerging Target Families: Intractable Targets. Handb Exp Pharmacol, 232 pp. 43-58. | Citations: 5 (European Pubmed Central) | Show Abstract | Read more

The druggability of a target is defined by the likelihood of a certain target binding site to be amendable to functional modulation by a small molecule in vivo. Thus, druggability depends on the ability of the developed small molecule to reach the target site, the properties of the ligand binding pocket and our ability to develop chemical matter that efficiently interact with the drug binding site of interest. Historically enzymes have been the main drug targets because the inhibition of their activity can be easily assayed and catalytic centres are often attractive drug binding sites. However, despite considerable effort, a number of classical enzyme families have not been successfully targeted. More recently protein-protein interactions received considerable attention and several clinical inhibitors have now been developed. Despite the considerable progress made expanding target space, a large number of targets with a very strong rationale for targeting remain intractable. In the following chapter I will summarize progress made in developing inhibitors for challenging drug binding sites and emerging target families.

Konopka T, Nijman SMB. 2016. Comparison of genetic variants in matched samples using thesaurus annotation. Bioinformatics, 32 (5), pp. 657-663. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

MOTIVATION: Calling changes in DNA, e.g. as a result of somatic events in cancer, requires analysis of multiple matched sequenced samples. Events in low-mappability regions of the human genome are difficult to encode in variant call files and have been under-reported as a result. However, they can be described accurately through thesaurus annotation-a technique that links multiple genomic loci together to explicate a single variant. RESULTS: We here describe software and benchmarks for using thesaurus annotation to detect point changes in DNA from matched samples. In benchmarks on matched normal/tumor samples we show that the technique can recover between five and ten percent more true events than conventional approaches, while strictly limiting false discovery and being fully consistent with popular variant analysis workflows. We also demonstrate the utility of the approach for analysis of de novo mutations in parents/child families. AVAILABILITY AND IMPLEMENTATION: Software performing thesaurus annotation is implemented in java; available in source code on github at GeneticThesaurus (https://github.com/tkonopka/GeneticThesaurus) and as an executable on sourceforge at geneticthesaurus (https://sourceforge.net/projects/geneticthesaurus). Mutation calling is implemented in an R package available on github at RGeneticThesaurus (https://github.com/tkonopka/RGeneticThesaurus). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: tomasz.konopka@ludwig.ox.ac.uk.

Ternette N, Yang H, Partridge T, Llano A, Cedeño S, Fischer R, Charles PD, Dudek NL, Mothe B, Crespo M et al. 2016. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells. Eur J Immunol, 46 (1), pp. 60-69. | Citations: 20 (Web of Science Lite) | Show Abstract | Read more

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T-cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4(+) T cells and the C8166 human T-cell line. Importantly, one-third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T-cell responses have previously been reported but for which the precise HLA class I-binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation during HIV-1 infection and provide novel targets for T-cell vaccine development.

Elkins JM, Fedele V, Szklarz M, Abdul Azeez KR, Salah E, Mikolajczyk J, Romanov S, Sepetov N, Huang X-P, Roth BL et al. 2016. Comprehensive characterization of the Published Kinase Inhibitor Set. Nat Biotechnol, 34 (1), pp. 95-103. | Citations: 83 (Scopus) | Show Abstract | Read more

Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome.

Morozumi Y, Boussouar F, Tan M, Chaikuad A, Jamshidikia M, Colak G, He H, Nie L, Petosa C, de Dieuleveult M et al. 2016. Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells. J Mol Cell Biol, 8 (4), pp. 349-362. | Citations: 15 (Scopus) | Show Abstract | Read more

Although the conserved AAA ATPase and bromodomain factor, ATAD2, has been described as a transcriptional co-activator upregulated in many cancers, its function remains poorly understood. Here, using a combination of ChIP-seq, ChIP-proteomics, and RNA-seq experiments in embryonic stem cells where Atad2 is normally highly expressed, we found that Atad2 is an abundant nucleosome-bound protein present on active genes, associated with chromatin remodelling, DNA replication, and DNA repair factors. A structural analysis of its bromodomain and subsequent investigations demonstrate that histone acetylation guides ATAD2 to chromatin, resulting in an overall increase of chromatin accessibility and histone dynamics, which is required for the proper activity of the highly expressed gene fraction of the genome. While in exponentially growing cells Atad2 appears dispensable for cell growth, in differentiating ES cells Atad2 becomes critical in sustaining specific gene expression programmes, controlling proliferation and differentiation. Altogether, this work defines Atad2 as a facilitator of general chromatin-templated activities such as transcription.

Bennett J, Fedorov O, Tallant C, Monteiro O, Meier J, Gamble V, Savitsky P, Nunez-Alonso GA, Haendler B, Rogers C et al. 2016. Discovery of a Chemical Tool Inhibitor Targeting the Bromodomains of TRIM24 and BRPF. J Med Chem, 59 (4), pp. 1642-1647. | Citations: 47 (Scopus) | Show Abstract | Read more

TRIM24 is a transcriptional regulator as well as an E3 ubiquitin ligase. It is overexpressed in diverse tumors, and high expression levels have been linked to poor prognosis in breast cancer patients. TRIM24 contains a PHD/bromodomain offering the opportunity to develop protein interaction inhibitors that target this protein interaction module. Here we identified potent acetyl-lysine mimetic benzimidazolones TRIM24 bromodomain inhibitors. The best compound of this series is a selective BRPF1B/TRIM24 dual inhibitor that bound with a KD of 137 and 222 nM, respectively, but exerted good selectivity over other bromodomains. Cellular activity of the inhibitor was demonstrated using FRAP assays as well as cell viability data.

Chen P, Chaikuad A, Bamborough P, Bantscheff M, Bountra C, Chung C-W, Fedorov O, Grandi P, Jung D, Lesniak R et al. 2016. Discovery and Characterization of GSK2801, a Selective Chemical Probe for the Bromodomains BAZ2A and BAZ2B. J Med Chem, 59 (4), pp. 1410-1424. | Citations: 65 (Scopus) | Show Abstract | Read more

Bromodomains are acetyl-lysine specific protein interaction domains that have recently emerged as a new target class for the development of inhibitors that modulate gene transcription. The two closely related bromodomain containing proteins BAZ2A and BAZ2B constitute the central scaffolding protein of the nucleolar remodeling complex (NoRC) that regulates the expression of noncoding RNAs. However, BAZ2 bromodomains have low predicted druggability and so far no selective inhibitors have been published. Here we report the development of GSK2801, a potent, selective and cell active acetyl-lysine competitive inhibitor of BAZ2A and BAZ2B bromodomains as well as the inactive control compound GSK8573. GSK2801 binds to BAZ2 bromodomains with dissociation constants (KD) of 136 and 257 nM for BAZ2B and BAZ2A, respectively. Crystal structures demonstrated a canonical acetyl-lysine competitive binding mode. Cellular activity was demonstrated using fluorescent recovery after photobleaching (FRAP) monitoring displacement of GFP-BAZ2A from acetylated chromatin. A pharmacokinetic study in mice showed that GSK2801 had reasonable in vivo exposure after oral dosing, with modest clearance and reasonable plasma stability. Thus, GSK2801 represents a versatile tool compound for cellular and in vivo studies to understand the role of BAZ2 bromodomains in chromatin biology.

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