Identification of PDE4B Over 4D subtype-selective inhibitors revealing an unprecedented binding mode.

A PDE4B over 4D-selective inhibitor programme was initiated to capitalise on the recently discovered predominance of the PDE4B subtype in inflammatory cell regulation. The SAR of a tetrahydrobenzothiophene (THBT) series did not agree with either of two proposed docking modes in the 4B binding site. A subsequent X-ray co-crystal structure determination revealed that the THBT ligand displaces the Gln-443 residue, invariably ligand-anchoring in previous PDE4 co-crystal structures, and even shifts helix-15 by 1-2A. For the first time, several residues of the C-terminus previously proposed to be involved in subtype selectivity are resolved and three of them extend into the ligand binding site potentially allowing for selective drug design.

Discovery and characterization of small molecule inhibitors of the BET family bromodomains.

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.

The integrin alphavbeta3 is a receptor for the latency-associated peptides of transforming growth factors beta1 and beta3.

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.

Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition.

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.