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Summary Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rβ1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rβ1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure., Graphical abstract, Highlights • NanoBRET assays were created to monitor the interaction of IL-23 with its receptor • The affinities of IL-23 for IL23R, IL12Rβ1 and heteromer complexes were measured • Receptor monomers were found to associate in complexes in the absence of IL-23 • IL-23 binding induced a rearrangement of the N-terminal domains of the receptor, Lay et al. measure the binding of TAMRA-labeled IL-23 to NanoLuciferase fused receptor subunits, revealing a high-affinity binding site formed from a heteromer of IL23R and IL12Rβ1. NanoBRET was also used to measure the interaction of subunits, demonstrating that IL-23 binding causes a conformational change of pre-formed receptor heteromers.

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB–POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB–POZ domain. The compound has good solubility (128 μg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

An ability to organize and encapsulate multiple active proteins into defined objects and spaces at the nanoscale has potential applications in biotechnology, nanotechnology, and synthetic biology. Previously, we have described the design, assembly, and characterization of peptide-based self-assembled cages (SAGEs). These ≈100 nm particles comprise thousands of copies of de novo designed peptide-based hubs that array into a hexagonal network and close to give caged structures. Here, we show that, when fused to the designed peptides, various natural proteins can be co-assembled into SAGE particles. We call these constructs pSAGE for protein-SAGE. These particles tolerate the incorporation of multiple copies of folded proteins fused to either the N or the C termini of the hubs, which modeling indicates form the external and internal surfaces of the particles, respectively. Up to 15% of the hubs can be functionalized without compromising the integrity of the pSAGEs. This corresponds to hundreds of copies giving mM local concentrations of protein in the particles. Moreover, and illustrating the modularity of the SAGE system, we show that multiple different proteins can be assembled simultaneously into the same particle. As the peptide-protein fusions are made via recombinant expression of synthetic genes, we envisage that pSAGE systems could be developed modularly to actively encapsulate or to present a wide variety of functional proteins, allowing them to be developed as nanoreactors through the immobilization of enzyme cascades or as vehicles for presenting whole antigenic proteins as synthetic vaccine platforms.

Activation of MrgX2, an orphan G protein‐coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine‐like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug‐like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug‐like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl‐Arg9‐amide) and TAT (49‐60), a fragment of HIV‐1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo‐allergic reactions known as red man syndrome, is an agonist of MrgX2.

The bromodomain of ATAD2 has proved to be one of the least-tractable proteins within this target class. Here, we describe the discovery of a new class of inhibitors by high-throughput screening and show how the difficulties encountered in establishing a screening triage capable of finding progressible hits were overcome by data-driven optimization. Despite the prevalence of nonspecific hits and an exceptionally low progressible hit rate (0.001%), our optimized hit qualification strategy employing orthogonal biophysical methods enabled us to identify a single active series. The compounds have a novel ATAD2 binding mode with noncanonical features including the displacement of all conserved water molecules within the active site and a halogen-bonding interaction. In addition to reporting this new series and preliminary structure-activity relationship, we demonstrate the value of diversity screening to complement the knowledge-based approach used in our previous ATAD2 work. We also exemplify tactics that can increase the chance of success when seeking new chemical starting points for novel and less-tractable targets.

Expression of mutant Huntingtin (HTT) protein is central to the pathophysiology of Huntington's Disease (HD). The E3 ubiquitin ligase MID1 appears to have a key role in facilitating translation of the mutant HTT mRNA suggesting that interference with the function of this complex could be an attractive therapeutic approach. Here we describe a peptide that is able to disrupt the interaction between MID1 and the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A). By fusing this peptide to a sequence from the HIV-TAT protein we demonstrate that the peptide can disrupt the interaction within cells and show that this results in a decrease in levels of ribosomal S6 phosphorylation and HTT expression in cultures of cerebellar granule neurones derived from HdhQ111/Q7 mice. This data serves to validate this pathway and paves the way for the discovery of small molecule inhibitors of this interaction as potential therapies for HD.

Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.

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

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.

Aims: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. Results: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. Innovation and Conclusions: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo. Antioxid. Redox Signal. 23, 358–374.

Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.

Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20 l scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.

A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997)Protein Eng. 10, 119–129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b 5. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b 5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochromeb 5. All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b 5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.

The extracellular interferon gamma receptor alpha-chain comprises two immunoglobulin-like domains, each with fibronectin type-III topology, which are responsible for binding interferon gamma at the cell surface. The epitopes on the human receptor recognized by three neutralizing antibodies, A6, gammaR38 and gammaR99, have been mapped by homolog scanning mutagenesis. In this way, a loop connecting beta-strands C and C' in the N-terminal domain was identified as a key component of the epitopes bound by A6 and gammaR38, whereas gammaR99 binds to the C-terminal domain in a region including strands A and B and part of the large C'E loop. The epitope for A6 was confirmed in a crystal structure of a complex between a recombinant N-terminal receptor domain and the Fab fragment from A6, determined by X-ray diffraction to 2.8 A resolution. The antibody-antigen interface buries 1662 A2 of protein surface, including 22 antibody residues from five complementarity determining regions, primarily through interactions with the CC' surface loop of the receptor. The floor of the antigen binding cavity is formed mainly by residues from CDR L3 and CDR H3 while a surrounding ridge is formed by residues from all other CDRs except L2. Many potential polar interactions, as well as 13 aromatic side-chains, four in VL, six in VH and three in the receptor, are situated at the interface. The surface of the receptor contacted by A6 overlaps to a large extent with that contacted by interferon-gamma, in the ligand-receptor complex. However, the conformation of this epitope is very different in the two complexes, demonstrating that conformational mobility in a surface loop on this cytokine receptor permits steric and electrostatic complementarity to two quite differently shaped binding sites.

Three single-chain antibody fragments that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R), and inhibit the binding of human IFN gamma, have been produced in Escherichia coli. These fragments are derived from murine anti-receptor monoclonal antibodies, and comprise the variable heavy (VH) domain linked to the variable light (VL) chain through a 15 amino acid linker [(GGGGS)3]. Using surface plasmon resonance technology (BIAcore), the soluble proteins were shown to retain a high affinity for recombinant IFN gamma R, and by radioimmunoassay to possess a high inhibitory activity towards IFN gamma-binding to human Raji cells. The antibody fragments most likely recognize epitopes that overlap the cytokine binding site on the receptor surface. Attempts to dissect further the antibodies to isolated VH- and VL-chains and to synthetic linear and cyclic peptides derived from the individual complementarity determining regions failed to afford fragments with significant IFN gamma R binding affinity. Nevertheless, these native-like variable region fragments and petidomimetics derived from them are of interest in the design of novel IFN gamma R antagonists.

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.

5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The optimization of the series to yield compounds with cell activity and brain permeability is described.

The discovery of a novel series of 2-(4-pyridyl)thienopyridinone GSK-3β inhibitors is reported. X-ray crystallography reveals its binding mode and enables rationalization of the SAR. The initial optimization of the template for improved cellular activity and predicted CNS penetration is also presented.

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.

We describe here a classical molecular modeling exercise that was carried out to provide a basis for the design of novel antagonist ligands of the CCR2 receptor. Using a theoretical model of the CCR2 receptor, docking studies were carried out to define plausible binding modes for the various known antagonist ligands, including our own series of indole piperidine compounds. On the basis of these results, a number of site-directed mutations (SDM) were designed that were intended to verify the proposed docking models. From these it was clear that further refinements would be necessary in the model. This was aided by the publication of a crystal structure of bovine rhodopsin, and a new receptor model was built by homology to this structure. This latest model enabled us to define ligand-docking hypotheses that were in complete agreement with the results of the SDM experiments.

Replying to B. Heinemann . 10.1038/nature13688 (2014) We welcome the accompanying Comment1 by Heinemann , in which the authors use an extensive panel of sensitive KDM assays to independently confirm our results2 that GSK-J1 is a potent KDM6 inhibitor. Additionally, Heinemann et al.1 demonstrate that GSK-J1 has some, albeit weaker, activity towards KDM5B and KDM5C, for which we only had preliminary data available at the time of our original publication. As our jumonji assay portfolio expands, we have continued to update the GSK-J1 activity profile on the SGC website ( http://www.thesgc.org/chemical-probes/GSKJ1 ); this includes KDM5 inhibition activity by GSK-J1 similar to that reported by Heinemann. In conclusion, GSK-J1 remains the most selective KDM inhibitor yet disclosed and thus a valuable chemical tool.

Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.

Background There are many challenges still to be overcome in the efficient production of recombinant protein from both eukaryotic and prokaryotic organisms. Often the problems faced are protein specific, and can result in unacceptable time delays between target screening and structural elucidation. A requirement therefore exists for rapid, parallel approaches that can identify suitable clones under a preliminary set of fermentation conditions in days rather than months.

A sleep questionnaire (SQ) was administered to 850 adults, 50 years and older, drawn from urban areas in 7 countries (Australia, Greece, Italy, Mexico, Spain, Venezuela, United States). All were functioning adults in relatively good physical and mental health. The samples were equated on sex, age, and occupational level. Of the 8 scales on the SQ, 7 showed statistically significant differences between countries, and 3 between males and females. At the same time, substantial similarities were found and are discussed from a normative point of view.

A 55-item Sleep Questionnaire is presented for possible use as a standardized instrument in psychological studies of sleep. A factor analysis of responses from 145 adults to the questionnaire indicated 7 factors accounted for 71.7% of the total variance. These factors are congruent with sleep dimensions discussed in the literature and with other factor analytic investigations. A set of 11 clinical judgment scales are also presented. The reliabilities of both factor and clinical scales are adequate as judged by test-retest, internal consistency, and comparisons of self vs spouse ratings. The construct validity is supported by three studies showing significant differences between (a) medical patients with and without sleep disturbances, (b) psychiatric patients with and without symptoms of depression, and (c) short and long sleepers.