Weiru Wang

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting “active” protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors.

Dynamic and clustering model of bacterial chemotaxis receptors: Structural basis for signaling and high sensitivity

Bacterial chemotaxis receptors can detect a small concentration gradient of attractants and repellents in the environment over a wide range of background concentration. The clustering of these receptors to form patches observed in vivo and in vitro has been suspected as a reason for the high sensitivity, and such wide dynamic range is thought to be due to the resetting of the receptor sensitivity threshold by methylation/demethylation of the receptors. However, the mechanisms by which such high sensitivity is achieved and how the methylation/demethylation resets the sensitivity are not well understood. A molecular modeling of an intact bacterial chemotaxis receptor based on the crystal structures of a cytoplasmic domain and a periplasmic domain suggests an interesting clustering of three dimeric receptors and a two-dimensional, close-packed lattice formation of the clusters, where each receptor dimer contacts two other receptor dimers at the cytoplasmic domain and two yet different receptor dimers at the periplasmic domain. This interconnection of the receptors to form a patch of receptor clusters suggests a structural basis for the high sensitivity of the bacterial chemotaxis receptors. Furthermore, we present crystallographic data suggesting that, in contrast to most molecular signaling by conformational changes and/or oligomerization of the signaling molecules, the changes in dynamic property of the receptors on ligand binding or methylation may be the language of the signaling by the chemotaxis receptors. Taken together, the changes of the dynamic property of one receptor propagating mechanically to many others in the receptor patch provides a plausible, simple mechanism for the high sensitivity and the dynamic range of the receptors.

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution.

BACKGROUND D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.

BeF(3)(-) acts as a phosphate analog in proteins phosphorylated on aspartate: structure of a BeF(3)(-) complex with phosphoserine phosphatase.

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

RAF inhibitors that evade paradoxical MAPK pathway activation

Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed ‘paradox breakers’) that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.

Scaffold-based discovery of indeglitazar, a PPAR pan-active anti-diabetic agent

In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARα, PPARγ, and PPARδ. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARγ, to circumvent the clinically observed side effects of full PPARγ agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARγ. Compared with full PPARγ-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).

Crystal structure of flavin binding to FAD synthetase of Thermotoga maritima.

Introduction. The product of genomic sequence gi:4981319 from Thermotoga maritima (TM379) is annotated as flavin adenine dinucleotide (FAD) synthetase, a bifunctional enzyme that catalyzes two reactions: riboflavin kinase (EC 2.7.1.26) and flavin mononucleotide (FMN) adenylyltransferase (EC 2.7.7.2). FAD synthetase is present among all kingdoms of living organisms. Mammals utilize separate enzymes for FMN and FAD formation, whereas lower level organisms depend on the bifunctional enzyme. The Berkeley Structural Genomics Center (BSGC) determined that the TM379 gene product possesses a novel fold and determined its crystal structure [Protein Data Bank (PDB) code: 1MRZ]. The TM379 structure exhibited a novel combination of two folds: a Rossmann fold at the N-terminus and a flavin binding fold at the C-terminus. While the sequence annotation is still awaiting experimental verification, our recently published structural data provided supportive evidence. Structural analysis suggests that the N-terminal domain of TM379 is involved in FMN adenylyltransferase, and the C-terminal domain contributes to binding of flavin and probably is involved in both steps of the reaction. Despite low sequence identity, the structural similarity between a recently published crystal structure of Schizosaccharomyces pombe (Sp) riboflavin kinase (RK) and the C-terminal domain of TM379 further indicated that the C-terminal domain is sufficient for catalyzing the RK reaction. The N-terminal domain also has structural similarity to nucleotidyltransferases, including glycerol-3-phosphate cytidylyltransferase (1COZ), nicotinamide mononucleotide adenylyltransferase (1F9A), and phosphopantetheine adenylyltransferase (1B6T), although sequence identities among them are low. To experimentally characterize the molecular function of TM397, we introduced riboflavin into TM379 crystal and determine the three-dimensional (3D) structure. Here we describe the cocrystal structure of TM379 and flavin, and identify the key residues involved in flavin binding for this family of proteins.

Crystal structure of tRNA (m1G37) methyltransferase from Aquifex aeolicus at 2.6 Å resolution: A novel methyltransferase fold

Introduction. The Berkeley Structural Genomics Center (BSGC) has focused on Mycoplasma as its structural genomics target organisms because of their compact genome size as well as their relevance to human and animal pathogenicity (http://www.strgen.org). The gene for transfer RNA (m1G37) methyltransferase of Aquifex aeolicus (GI number 2983865) is one of the structural genomics targets of BSGC that has been selected as a homolog of the Mycoplasma pneumoniae (MP) gene MPN183, trmD (http:// www.strgen.org/status/mptargets.html). The protein structures coded by either gene are not known. It is one of the tRNA-modifying enzymes that catalyzes the transfer of methyl group from S-adenosyl-L-methionine (AdoMet) to guanosine at position 37, the nucleoside adjacent to and 3 of the anticodon. This protein is important for the maintenance of the correct reading frame during translation. In all organisms, the tRNA-reading codons CUN (Leu), CCN (Pro), and CGG (Arg) contain at position 37 1-methylguanosine (m1G37), and tRNA (m1G37) methyltransferases from members of all three phylogenetic domains show sequence similarities. Currently, no structural information is available for this protein family. Here, we report the crystal structure of the enzyme tRNA (m1G37) methyltransferase from Aquifex aeolicus at 2.6 Å resolution. The structure reveals a novel methyltransferase fold distinctly different from those of the most common methyltransferases.

Crystal structure of a flavin-binding protein from Thermotoga maritima.

. TheTM379 structure is composed of two structural domainswith a single loop linkage in between. The N-terminaldomain (residues 2–134) adopts a typical nucleotide-binding fold (Rossmann fold). The C-terminal domain(residues 135–288) contains a six-stranded antiparallelBluntII-TOPO vector (Invitrogen) and the TM379 gene-barrel architecture. The C-terminal domain also in-cludes three -helices. Two of them are on the N-terminalend and one long helix is on the C-terminal end. The twoN-terminalhelicesarelocatedatthebottomofthe -barrelshown in Figure 1. The C-terminal helix runs across theside of the -barrel and is oriented approximately perpen-dicular to the -barrel’s axis.Structural homology search using DALI

Chemotaxis Receptor in Bacteria

Publisher Summary This chapter provides an understanding of the chemotaxis receptor in bacteria. Chemotaxis is the phenomenon in which somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements according to certain chemicals in their environment. Bacteria rapidly respond to changes in concentrations of critical chemicals in their environment by chemotaxis—that is, a swimming pattern biased toward or away from particular stimuli. The chemotaxis pathway includes chemosensory receptors and a phosphotransfer system known as the two-component signal transduction pathway. Most bacterial chemoreceptors belong to a family of transmembrane methyl-accepting chemotaxis proteins (MCPs). This study begins by describing signaling at the periplasmic ligand binding domain, using the examples of Salmonella tryphimurium aspartate receptor (Tar) mutant in apo and liganded (Asp bound) forms and the wild-type Tar of apo and liganded forms. Ligand binding induces small conformational changes, which are transmitted through the transmembrane helices to the cytoplasmic domain and affect the phosphorylation rate of the bound histidine kinase. Following this, the study provides an understanding of signaling in the cytoplasmic domain, using the example of E coli serine receptor (Tsr), Q mutant. Furthermore, it describes adaptation of the chemotaxis pathway, which enables the cell to adapt to a constant background stimulus so that it can chemotax up a small concentration gradient superimposed on a large background level of attractant. Finally, it deals with clustering of the chemoreceptor and sensitivity.