Lipid-Dependent Activation of the Orphan G Protein-Coupled Receptor, GPR3.

The class A orphan G protein-coupled receptor (GPCR), GPR3, has been implicated in a variety of conditions, including Alzheimer's and premature ovarian failure. GPR3 constitutively couples with Gαs, resulting in the production of cAMP in cells. While tool compounds and several putative endogenous ligands have emerged for the receptor, its endogenous ligand, if it exists, remains a mystery. As novel potential drug targets, the structures of orphan GPCRs have been of increasing interest, revealing distinct modes of activation, including autoactivation, presence of constitutively activating mutations, or via cryptic ligands. Here, we present a cryo-electron microscopy (cryo-EM) structure of the orphan GPCR, GPR3 in complex with DNGαs and Gß1γ2. The structure revealed clear density for a lipid-like ligand that bound within an extended hydrophobic groove, suggesting that the observed "constitutive activity" was likely due to activation via a lipid that may be ubiquitously present. Analysis of conformational variance within the cryo-EM data set revealed twisting motions of the GPR3 transmembrane helices that appeared coordinated with changes in the lipid-like density. We propose a mechanism for the binding of a lipid to its putative orthosteric binding pocket linked to the GPR3 dynamics.

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using single-molecule techniques, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-piconewton mechanical forces. Stepwise decondensation at high force and DNA braiding experiments show that the condensation activity is due to the stabilization of DNA loops by PARP1. PARP inhibitors do not affect the level of condensation of undamaged DNA but act to block condensation reversal for damaged DNA in the presence of NAD+ Our findings suggest a mechanism for PARP1 in the organization of chromatin structure.

High-throughput virtual screening of proteins using GRID molecular interaction fields.

A new computational algorithm for protein binding sites characterization and comparison has been developed, which uses a common reference framework of the projected ligand-space four-point pharmacophore fingerprints, includes cavity shape, and can be used with diverse proteins as no structural alignment is required. Protein binding sites are first described using GRID molecular interaction fields (GRID-MIFs), and the FLAP (fingerprints for ligands and proteins) method is then used to encode and compare this information. The discriminating power of the algorithm and its applicability for large-scale protein analysis was validated by analyzing various scenarios: clustering of kinase protein families in a relevant manner, predicting ligand activity across related targets, and protein-protein virtual screening. In all cases the results showed the effectiveness of the GRID-FLAP method and its potential use in applications such as identifying selectivity targets and tools/hits for new targets via the identification of other proteins with pharmacophorically similar binding sites.

Fragment-based drug discovery: what has it achieved so far?

Fragment-based drug discovery has proved to be a very useful approach particularly in the hit-to-lead process, providing a complementary tool to traditional high-throughput screening. Although often synonymous with fragment screening, fragment-based drug discovery is a far wider area covering high-throughput screening, fragment screening and virtual screening efforts. The unifying feature of fragment-based drug discovery is the low molecular weight of the hit rather than the approach it originates from. Over the last ten years, fragment-based drug discovery has provided in excess of 50 examples of small molecule hits that have been successfully advanced to leads and therefore resulted in useful substrate for drug discovery programs. To our knowledge, there are currently no marketed drugs that can be attributed to these efforts. However, due to the time scales of drug discovery and development it is likely that over the next few years the number of such examples will increase significantly.

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.