Transcriptional regulation by the glucocorticoid receptor (GR) is mediated by hormone binding, receptor dimerization, and coactivator recruitment. Here, we report the crystal structure of the human GR ligand binding domain (LBD) bound to dexamethasone and a coactivator motif derived from the transcriptional intermediary factor 2. Despite structural similarity to other steroid receptors, the GR LBD adopts a surprising dimer configuration involving formation of an intermolecular beta sheet. Functional studies demonstrate that the novel dimer interface is important for GR-mediated activation. The structure also reveals an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand binding pocket that explains its selectivity for endogenous steroid hormones. These results establish a framework for understanding the roles of protein-hormone and protein-protein interactions in GR signaling pathways.

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.

These structures show how two drugs interact with a fungal DHFR. A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species. These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.

The integrins alpha(v)beta(1), alpha(v)beta(5), alpha(v)beta(6) and alpha(v)beta(8) have all recently been shown to interact with the RGD motif of the latency-associated peptide (LAPbeta(1)) of transforming growth factor beta(1) (TGFbeta(1)), with binding to alpha(v)beta(6) and alpha(v)beta(8) leading to TGFbeta(1) activation. Previously it has been suggested that the remaining alpha(v) integrin, alpha(v)beta(3,) does not interact with LAPbeta(1). However, here we show clearly that alpha(v)beta(3) does indeed interact with the LAPbeta(1) RGD motif. This interaction is similar to other alpha(v)beta(3) ligands in terms of the cations required for adhesion, the concentrations of LAPbeta(1) required for binding and the ability of a small-molecule inhibitor of alpha(v)beta(3), SB223245, to block the interaction. Using glutathione S-transferase fusion proteins we have mapped a minimal integrin-binding loop in LAPbeta(1) and then used this approach to probe the integrin-binding properties of the equivalent loops in LAPbeta(2) and LAPbeta(3). We show that the RGD motif of LAPbeta(3) also interacts with alpha(v)beta(3), in addition to alpha(v)beta(6), alpha(v)beta(1) and alpha(v)beta(5), whereas the corresponding loop in LAPbeta(2) does not interact with these integrins. These observations therefore correct a previously reported inaccuracy in the literature. Furthermore, they are important as they link alpha(v)beta(3) and TGFbeta, which may have implications in cancer and a number of inflammatory and fibrotic diseases where expression of both proteins has been documented.

The nucleotide sequence of a folk acid synthesis (fax) gene from Pneumocystis curinii contains an open reading frame (ORF) that predicts a protein of 740 amino acids with an M, of 83979. A recombinant baculovirus was constructed which directed expression of the predicted Fas740 polypeptide in cultured Spodoptera frugiperdu (SF9) insect cells. The overexpressed 'full-length' protein migrated anomalously in sodium dodecyl sulfate/polyacrylamide gels, with an apparent molecular mass of 71.5 kDa. An abundant 69-kDa species was also recognized by polyclonal sera specific for the Fas protein in immunoblotting analyses. Dihydroneopterin aldolase, dihydropterin pyrophosphokinase and dihydropteroate synthase activities were readily detected in SF9 extracts in which the 71.5/69-kDa immunoreactive species were overproduced, demonstrating that three enzyme functions involved in catalysing three sequential steps of the folate biosynthetic pathway are encoded by a single gene in R curinii. Importantly, the polyclonal sera recognize a single 69-kDa species in R curinii extracts suggesting that the three activities are indeed properties of a single polypeptide, although the nature of the suggested post-translational modification is unknown. Location of the individual enzyme domains with the Fas polypeptide based upon amino acid sequence similarity to their bacterial counterparts is discussed. Futhermore, expression of various truncated fus gene constructs demonstrates that the complete fus ORF, including the N-terminus of the predicted polypeptide (FasA domain) whose enzyme function is unknown, must be expressed for maximum dihydroneopterin aldolase (FasB domain) and dihydropteroate synthase (FasD domain) activites. This suggests interactions between the domains within the larger polypeptide to stabilize the functions of these two enzymes. The FasC domain, which contains 6-hydroxymethyl-7,s-dihydropterin pyrophosphokinase activity, is able to fold and function independently of the other domains. The requirement by mammalian cells for preformed folates, and the absence of dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and dihydropteroate synthase from these tissues opens up the possibility of designing higly selective drugs which inhibit these unique targets.Reduced folate cofactors are essential for the syntheses of purines, thymidylate, glycine, methionine, pantothenic acid, and N-formylmethionyl-tRNA in all cells [ 11. Most microbes must synthesise folates de n o w since they lack the Correspondence to C . J.

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Activated T cells drive a range of immune-mediated inflammatory diseases. LAG-3 is transiently expressed on recently activated CD4 + and CD8 + T cells. We describe the engineering and first-inhuman clinical study (NCT02195349) of GSK2831781 (an afucosylated humanized IgG1 monoclonal antibody enhanced with high affinity for Fc receptors and LAG-3 and antibody-dependent cellular cytotoxicity capabilities), which depletes LAG-3 expressing cells. GSK2831781 was tested in a phase I/Ib, double-blind, placebo-controlled clinical study, which randomized 40 healthy participants (part A) and 27 patients with psoriasis (part B) to single doses of GSK2831781 (up to 0.15 and 5 mg/kg, respectively) or placebo. Adverse events were generally balanced across groups, with no safety or tolerability concern identified. LAG-3 + cell depletion in peripheral blood was observed at doses ≥ 0.15 mg/ kg and was dose-dependent. In biopsies of psoriasis plaques, a reduction in mean group LAG-3 + and CD3 + T-cell counts was observed following treatment. Downregulation of proinflammatory genes (IL-17A, IL-17F, IFNγ, and S100A12) and upregulation of the epithelial barrier integrity gene, CDHR1, was observed with the 5 mg/kg dose of GSK2831781. Psoriasis disease activity improved up to day 43 at all GSK2831781 doses (0.5, 1.5, and 5 mg/kg) compared with placebo. Depletion of LAG-3-expressing activated T cells is a novel approach, and this first clinical study shows that GSK2831781 is pharmacologically active and provides encouraging early evidence of clinical effects in psoriasis, which warrants further investigation in T-cell-mediated inflammatory diseases.