40 results on '"Robert T. Nolte"'
Search Results
2. Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase.
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Kushol Gupta, Vesa Turkki, Scott Sherrill-Mix, Young Hwang, Grant Eilers, Louis Taylor, Charlene McDanal, Ping Wang, David Temelkoff, Robert T Nolte, Emile Velthuisen, Jerry Jeffrey, Gregory D Van Duyne, and Frederic D Bushman
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Biology (General) ,QH301-705.5 - Abstract
The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.
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- 2016
- Full Text
- View/download PDF
3. BacMam production and crystal structure of nonglycosylated apo human furin at 1.89 Å resolution
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Laurie K. Overton, J. David Taylor, D.D. McKee, Nino Campobasso, Robert T. Nolte, Robert A. Reid, Kenneth H. Pearce, George B. Barrett, and Robert T. Gampe
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Glycosylation ,animal structures ,viruses ,Biophysics ,BacMam ,CHO Cells ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Research Communications ,Serine ,03 medical and health sciences ,chemistry.chemical_compound ,Cricetulus ,0302 clinical medicine ,Protein Domains ,Structural Biology ,Cricetinae ,Genetics ,Animals ,Humans ,Amino Acid Sequence ,Furin ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,fungi ,Subtilisin ,Condensed Matter Physics ,Proprotein convertase ,Cell biology ,HEK293 Cells ,Ectodomain ,030220 oncology & carcinogenesis ,embryonic structures ,biology.protein ,Kexin ,Apoproteins - Abstract
Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.
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- 2019
4. Dolutegravir interactions with HIV-1 integrase-DNA: structural rationale for drug resistance and dissociation kinetics.
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Felix DeAnda, Kendra E Hightower, Robert T Nolte, Kazunari Hattori, Tomokazu Yoshinaga, Takashi Kawasuji, and Mark R Underwood
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Medicine ,Science - Abstract
Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir's antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir's prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.
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- 2013
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5. A knowledge-based, structural-aided discovery of a novel class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors
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Young Do, Lisa A. Orband-Miller, Anthony Shillings, Joelle Le, Caterina Musetti, Gordon Saxty, Beth Pietrak, Simon Teanby Hodgson, Petrov Kimberly, Daniel J. Price, Stephen A. Thomson, Eugene L. Stewart, Christie Schulte, Ashley Paul Hancock, Terrence L. Smalley, Michael R. Jeune, H. Fritz Kramer, Chuck Poole, Heather Hobbs, Kirsten M. Kahler, J. Darren Stuart, Paul N. Mortenson, Robert T. Nolte, Jason A. Holt, David N. Deaton, Don O. Somers, Elsie Diaz, Jeffrey Guss, Robert T. Gampe, and Peckham Gregory
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Imidazopyridine ,medicine.drug_class ,Stereochemistry ,Clinical Biochemistry ,Pharmaceutical Science ,Carboxamide ,01 natural sciences ,Biochemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Discovery ,Pyridine ,Virtual screen ,medicine ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Class (computer programming) ,Dose-Response Relationship, Drug ,Molecular Structure ,010405 organic chemistry ,Organic Chemistry ,0104 chemical sciences ,Intramolecular Oxidoreductases ,010404 medicinal & biomolecular chemistry ,Orally active ,chemistry ,Molecular Medicine - Abstract
Through an internal virtual screen at GlaxoSmithKline a distinct class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors were discovered. Careful evaluation of crystal structures and SAR led to a novel, potent, and orally active imidazopyridine inhibitor of H-PGDS, 20b. Herein, describes the identification of 2 classes of inhibitors, their syntheses, and their challenges.
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- 2021
6. The Role of Phosphodiesterase 12 (PDE12) as a Negative Regulator of the Innate Immune Response and the Discovery of Antiviral Inhibitors
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Liping Wang, Shihyun You, Ninad V. Prabhu, Hongfeng Deng, Danielle G. Smith, Jing Chai, Ginger H Tomberlin, Robert T. Nolte, James H. Nichols, G. Bruce Wisely, Edgar R. Wood, Cecil E Rise, Hamilton D. Dickson, J. David Taylor, Luz Helena Kryn, Randy K. Bledsoe, Timothy P. Sheahan, Yun Ding, Sarah Harris-Gurley, Eldridge N. Nartey, J. Brad Shotwell, Bing Xia, Philias Daka, and Vince Tai
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Models, Molecular ,Rhinovirus ,Biology ,Crystallography, X-Ray ,Biochemistry ,Antiviral Agents ,Virus ,Small Molecule Libraries ,Gene Knockout Techniques ,Structure-Activity Relationship ,Interferon ,Transcription (biology) ,Endoribonucleases ,medicine ,2',5'-Oligoadenylate Synthetase ,Escherichia coli ,Humans ,Encephalomyocarditis virus ,Molecular Biology ,Regulation of gene expression ,Innate immune system ,Oligoribonucleotides ,Effector ,Adenine Nucleotides ,Phosphodiesterase ,Interferon-alpha ,Molecular Bases of Disease ,Cell Biology ,Virology ,Immunity, Innate ,Recombinant Proteins ,Respiratory Syncytial Viruses ,Poly I-C ,Gene Expression Regulation ,Cell culture ,Exoribonucleases ,medicine.drug ,HeLa Cells ,Signal Transduction - Abstract
2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.
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- 2015
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7. Allosteric Inhibition of Human Immunodeficiency Virus Integrase
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Troy Brady, Benjamin M. Dyer, Emile Johann Velthuisen, Nirav Malani, Kushol Gupta, Jerry Jeffrey, Gregory D. Van Duyne, Young Hwang, Liping Wang, Frances Male, Robert T. Nolte, and Frederic D. Bushman
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biology ,Viral protein ,Allosteric regulation ,Cell Biology ,biology.organism_classification ,medicine.disease_cause ,Biochemistry ,Molecular biology ,Integrase ,Retrovirus ,Mechanism of action ,Viral replication ,Cell culture ,medicine ,biology.protein ,medicine.symptom ,Molecular Biology ,Transcription factor - Abstract
HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action.
- Published
- 2014
8. Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase
- Author
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Robert T. Nolte, Louis J. Taylor, Scott Sherrill-Mix, Gregory D. Van Duyne, Jerry Jeffrey, Young Hwang, Charlene B. McDanal, Kushol Gupta, Grant Eilers, David Temelkoff, Ping Wang, Vesa Turkki, Emile Johann Velthuisen, and Frederic D. Bushman
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0301 basic medicine ,RNA viruses ,Polymers ,Mutant ,HIV Integrase ,Pathology and Laboratory Medicine ,Immunodeficiency Viruses ,Medicine and Health Sciences ,Biology (General) ,chemistry.chemical_classification ,Crystallography ,Molecular Structure ,General Neuroscience ,Physics ,Condensed Matter Physics ,3. Good health ,Integrase ,Amino acid ,Chemistry ,Biochemistry ,Macromolecules ,Medical Microbiology ,Viral Pathogens ,Viruses ,Physical Sciences ,Crystal Structure ,Pathogens ,General Agricultural and Biological Sciences ,Research Article ,Substitution Mutation ,QH301-705.5 ,Materials by Structure ,Chemical physics ,Allosteric regulation ,Materials Science ,Biology ,Viral Structure ,Microbiology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Allosteric Regulation ,Virology ,Retroviruses ,Genetics ,Viral Core ,Solid State Physics ,DNA Integration ,HIV Integrase Inhibitors ,Microbial Pathogens ,General Immunology and Microbiology ,Point mutation ,Lentivirus ,Organisms ,Biology and Life Sciences ,HIV ,Dimers (Chemical physics) ,Polymer Chemistry ,Viral Replication ,030104 developmental biology ,Viral replication ,chemistry ,Mutation ,biology.protein ,Biophysics ,HIV-1 ,CTD - Abstract
The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization., A new crystal structure of the HIV integrase enzyme in complex with the allosteric inhibitor GSK1264 explains how the drug induces aggregation of the viral protein., Author Summary A promising new class of antivirals called “ALLINIs” (allosteric inhibitors of integrase) potently inhibits HIV replication. Like other drugs, ALLINIs seem to target also the HIV-1 integrase (IN), which is crucial for the replication of this virus, but instead of acting at early phases of HIV replication, they interfere with viral particle assembly and maturation that occur at late stages and induce aggregation of IN. Despite these findings, the structural bases for the effects are still unknown. In this study, we crystallized full-length HIV-1 IN in complex with an ALLINI called GSK1264 and determined its structure to 4.4 Å. The structure reveals for the first time the complete ALLINI-binding interface, comprised of both IN C-terminal and catalytic core domains. These domains are contributed from neighboring IN dimers, revealing an open polymeric conformation mediated by inhibitor-bridged contacts. Substitutions at this interface block ALLINI-induced multimerization, and we find that escape mutants against this class of drug lie at or near this interface. We propose that ALLINIs catalyze formation of an open IN polymer, which in turn interferes with viral particle assembly.
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- 2016
9. Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop
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Oliver S. Smart, Paul Emsley, Cary B. Bauer, David A. Case, John L. Markley, Joseph Marcotrigiano, Jasmine Young, Atsushi Nakagawa, Seth F. Harris, Haruki Nakamura, Wolfram Tempel, Radka Svobodová, T. Krojer, Pamela A. Williams, Robert T. Nolte, Catherine E. Peishoff, Jorg Hendle, Chenghua Shao, Jeff Blaney, Dale E. Tronrud, Paul D. Adams, Randy J. Read, Marc C. Nicklaus, Kirk Clark, Helen M. Berman, Jeffrey A. Bell, Evan E Bolton, Suzanna C. Ward, Stephen K. Burley, Alan E. Mark, Garib N. Murshudov, Victoria A. Feher, Matthew T. Miller, John Spurlino, Sameer Velankar, Steven Sheriff, Tom Darden, Wladek Minor, Talapady N. Bhat, John D. Westbrook, Gerard J. Kleywegt, Terry R. Stouch, Huanwang Yang, Gérard Bricogne, Thomas C. Terwilliger, Anil K. Padyana, Zukang Feng, Colin R. Groom, Andrzej Joachimiak, David G. Brown, Anthony Nicholls, Gaetano T. Montelione, Thomas Holder, Kathleen Aertgeerts, Stephen M. Soisson, Gregory L. Warren, Susan Pieniazek, Read, Randy [0000-0001-8273-0047], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Models, Molecular ,Protein Conformation ,Complex formation ,Protein Data Bank (RCSB PDB) ,Biophysics ,Crystallographic data ,Guidelines as Topic ,010402 general chemistry ,Crystallography, X-Ray ,Ligands ,01 natural sciences ,Article ,03 medical and health sciences ,Structural bioinformatics ,Databases ,Extant taxon ,Structural Biology ,Models ,Information and Computing Sciences ,Databases, Protein ,Molecular Biology ,Data Curation ,Crystallography ,Ligand ,Chemistry ,Protein ,Molecular ,Proteins ,computer.file_format ,Collaboratory ,Biological Sciences ,Protein Data Bank ,Data science ,0104 chemical sciences ,030104 developmental biology ,Generic Health Relevance ,QD431 ,Chemical Sciences ,X-Ray ,computer - Abstract
Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent\ud an important source of information concerning drug-target interactions, providing atomic level insights\ud into the physical chemistry of complex formation between macromolecules and ligands. Of the\ud more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ~75% include at least one non-polymeric\ud ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug\ud discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across\ud the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic\ud Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop\ud held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30–31, 2015.\ud Experts in protein crystallography from academe and industry came together with non-profit and for-profit\ud software providers for crystallography and with experts in computational chemistry and data archiving to\ud discuss and make recommendations on best practices, as framed by a series of questions central to structural\ud studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived\ud in the PDB? How should the ligands be best represented? How should structural models of macromoleculeligand\ud complexes be validated? What supplementary information should accompany publications of structural\ud studies of biological macromolecules? Consensus recommendations on best practices developed in\ud response to each of these questions are provided, together with some details regarding implementation.\ud Important issues addressed but not resolved at the workshop are also enumerated.
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- 2016
10. Rational Design of Potent Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase
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Andrew J. Peat, Kurt Weaver, Liping Wang, Sebahar Paul Richard, Eugene L. Stewart, Amanda Mathis, Robert T. Nolte, Dulce Garrido, Robert G. Ferris, Mark P. Edelstein, Huichang Zhang, Pek Yoke Chong, and Michael Youngman
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Models, Molecular ,Chemistry ,Rational design ,Human immunodeficiency virus (HIV) ,Microbial Sensitivity Tests ,Crystallography, X-Ray ,medicine.disease_cause ,Virology ,HIV Reverse Transcriptase ,Reverse transcriptase ,Biochemistry ,Drug Design ,Drug Discovery ,Hydrolase ,HIV-1 ,medicine ,Reverse Transcriptase Inhibitors ,Molecular Medicine ,Nucleoside - Abstract
A new series of non-nucleoside reverse transcriptase inhibitors based on an imidazole-amide biarylether scaffold has been identified and shown to possess potent antiviral activity against HIV-1, including the NNRTI-resistant Y188L mutated virus. X-ray crystallography of inhibitors bound to reverse transcriptase, including a structure of the Y188L RT protein, was used extensively to help identify and optimize the key hydrogen-bonding motif. This led directly to the design of compound 43 that exhibits remarkable antiviral activity (EC501 nM) against a wide range of NNRTI-resistant viruses and a favorable pharmacokinetic profile across multiple species.
- Published
- 2012
11. Structure of Rev-erbα bound to N-CoR reveals a unique mechanism of nuclear receptor–co-repressor interaction
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Valerie G. Montana, Robert T. Nolte, Jane Bynum, Robert T. Gampe, Xiao Hu, Derek J. Parks, Mitchell A. Lazar, Shawn P. Williams, Millard H. Lambert, Timothy Broderick, and Caroline A. Phelan
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Nuclear cap-binding protein complex ,Peptide binding ,Biology ,Molecular biology ,chemistry.chemical_compound ,Protein structure ,chemistry ,Structural Biology ,Biophysics ,Molecular Biology ,Heme ,Psychological repression ,Peptide sequence ,Protein secondary structure ,Nuclear receptor co-repressor 1 - Abstract
Repression of gene transcription by the nuclear receptor Rev-erbalpha plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor-co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbalpha ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel beta-sheet with Rev-erbalpha, as well as an alpha-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbbeta bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbalpha could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor-co-repressor interactions.
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- 2010
12. Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes: 1. Identification of 1-amino-1-cycloalkyl carboxylic acid headgroups
- Author
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Pierette Banker, Daphne C. Clancy, Francis X. Tavares, Pamela L. Golden, Liping Wang, James E. Weiel, Steven M. Sparks, Stephen A. Thomson, Lauren R. Sheckler, Dulce Garrido, Scott Howard Dickerson, Andrew J. Peat, David M. Bickett, H. Luke Carter, Kate A. Dwornik, and Robert T. Nolte
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Stereochemistry ,Chemistry, Pharmaceutical ,Carboxylic acid ,Clinical Biochemistry ,Carboxylic Acids ,Glycine ,Molecular Conformation ,Pharmaceutical Science ,Crystallography, X-Ray ,Imides ,Biochemistry ,Inhibitory Concentration 50 ,chemistry.chemical_compound ,Glycogen phosphorylase ,Dogs ,Drug Discovery ,Animals ,Humans ,ortho-Aminobenzoates ,Glycogen synthase ,Phosphorylase kinase ,Molecular Biology ,Cytochrome P-450 CYP2C9 ,chemistry.chemical_classification ,Glycogen ,biology ,Glycogen Phosphorylase ,Organic Chemistry ,Rats ,Amino acid ,Enzyme ,Diabetes Mellitus, Type 2 ,Liver ,chemistry ,Enzyme inhibitor ,Drug Design ,biology.protein ,Molecular Medicine ,Aryl Hydrocarbon Hydroxylases - Abstract
Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.
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- 2009
13. Discovery of Novel Benzimidazoles as Potent Inhibitors of TIE-2 and VEGFR-2 Tyrosine Kinase Receptors
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Robert T. Nolte, Kazuya Kano, Liping Wang, James Marvin Veal, Philip A. Harris, Hideyuki Sato, Masaichi Hasegawa, Rob I. GlaxoSmithKline West, Megumi Shibahara, Yoshiaki Washio, Mui Cheung, Naohiko GlaxoSmithKline Nishigaki, Hiroko Toyoda, and Ichiro Mori
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Models, Molecular ,Benzimidazole ,Stereochemistry ,Angiogenesis ,Substituent ,Crystallography, X-Ray ,Receptor tyrosine kinase ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Discovery ,Animals ,Humans ,Moiety ,Phosphorylation ,Receptor ,chemistry.chemical_classification ,Binding Sites ,Molecular Structure ,biology ,Kinase ,Receptor, TIE-2 ,Vascular Endothelial Growth Factor Receptor-2 ,Enzyme ,chemistry ,Biochemistry ,NIH 3T3 Cells ,biology.protein ,Molecular Medicine ,Benzimidazoles - Abstract
We herein disclose a novel chemical series of benzimidazole-ureas as inhibitors of VEGFR-2 and TIE-2 kinase receptors, both of which are implicated in angiogenesis. Structure-activity relationship (SAR) studies elucidated a critical role for the N1 nitrogen of both the benzimidazole (segment E) and urea (segment B) moieties. The SAR results were also supported by the X-ray crystallographic elucidation of the role of the N1 nitrogen and the urea moiety when the benzimidazole-urea compounds were bound to the VEGFR-2 enzyme. The left side phenyl ring (segment A) occupies the backpocket where a 3-hydrophobic substituent was favored for TIE-2 activity.
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- 2007
14. Co-crystal structure guided array synthesis of PPARγ inverse agonists
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Marie A. Iannone, Lisa M. Leesnitzer, Andrew N. Billin, Timothy M. Willson, Kenneth H. Pearce, Robert T. Nolte, William J. Hoekstra, Sumin M. Zhao, Richard G. Buckholz, Millard H. Lambert, Barry G. Shearer, Cobb Jeffrey Edmond, and Ryan P. Trump
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Magnetic Resonance Spectroscopy ,Molecular model ,medicine.drug_class ,Carboxylic acid ,Clinical Biochemistry ,Pharmaceutical Science ,Adipose tissue ,Carboxamide ,Ligands ,Biochemistry ,Chemical synthesis ,Mass Spectrometry ,Farglitazar ,Drug Discovery ,medicine ,Inverse agonist ,Oxazoles ,Molecular Biology ,chemistry.chemical_classification ,Molecular Structure ,Organic Chemistry ,PPAR gamma ,chemistry ,Nuclear receptor ,Tyrosine ,Molecular Medicine ,Crystallization ,medicine.drug - Abstract
PPARgamma-activating thiazolidinediones and carboxylic acids such as farglitazar exert their anti-diabetic effects in part in PPARgamma rich adipose. Both pro- and anti-adipogenic PPARgamma ligands promote glucose and lipid lowering in animal models of diabetes. Herein, we disclose representatives of an array of 160 farglitazar analogues with atypical inverse agonism of PPARgamma in mature adipocytes.
- Published
- 2007
15. Orally active 4-amino-5-diarylurea-furo[2,3-d]pyrimidine derivatives as anti-angiogenic agent inhibiting VEGFR2 and Tie-2
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Daniel F. Hassler, Robert T. Nolte, Denis Patrick, Jun Tang, Masato Nakano, Kazunori Ozawa, Maureen L. Ho, Eldridge N. Nartey, Yasushi Miyazaki, Yutaka Maeda, Hideyuki Sato, Anne T. Truesdale, Masaki Sugai, Liping Wang, and Yuji Okamoto
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Male ,Models, Molecular ,Pyrimidine ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Pharmaceutical Science ,Angiogenesis Inhibitors ,Antineoplastic Agents ,Pharmacology ,Crystallography, X-Ray ,Biochemistry ,Chemical synthesis ,Mice ,chemistry.chemical_compound ,Pharmacokinetics ,Growth factor receptor ,Oral administration ,Drug Discovery ,Animals ,Humans ,Urea ,Computer Simulation ,Molecular Biology ,chemistry.chemical_classification ,Trifluoromethyl ,Organic Chemistry ,Biological activity ,Receptor, TIE-2 ,Vascular Endothelial Growth Factor Receptor-2 ,Pyrimidines ,Enzyme ,chemistry ,Area Under Curve ,RNA ,Molecular Medicine ,Female ,Indicators and Reagents ,HT29 Cells ,Neoplasm Transplantation - Abstract
During our effort to develop dual VEGFR2 and Tie-2 inhibitors as anti-angiogenic agents for cancer therapy, we discovered 4-amino-5-(4-((2-fluoro-5-(trifluoromethyl)phenyl)- aminocarbonylamino)phenyl)furo[2,3-d]pyrimidine (8a) possessing strong inhibitory activity at both the enzyme and cellular level against VEGFR2 and Tie-2. Compound 8a demonstrated high pharmacokinetic exposure through oral administration, and showed marked tumor growth inhibition and anti-angiogenic activity in mouse HT-29 xenograft model via once-daily oral administration.
- Published
- 2007
16. Structure-guided synthesis of tamoxifen analogs with improved selectivity for the orphan ERRγ
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Timothy M. Willson, William J. Zuercher, Robert T. Nolte, Lisa A. Orband-Miller, Jon L. Collins, Aaron B. Miller, Donald P. McDonnell, Esther Y. Chao, Liping Wang, and Stephanie Gaillard
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Models, Molecular ,Stereochemistry ,Clinical Biochemistry ,Receptors, Cytoplasmic and Nuclear ,Pharmaceutical Science ,Estrogen receptor ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Chemical synthesis ,Structure-Activity Relationship ,Drug Discovery ,Humans ,Inverse agonist ,Receptor ,Molecular Biology ,Binding selectivity ,Orphan receptor ,Binding Sites ,Molecular Structure ,Chemistry ,Organic Chemistry ,Estrogen Receptor alpha ,Tamoxifen ,Receptors, Estrogen ,Nuclear receptor ,Drug Design ,Molecular Medicine ,Selectivity ,hormones, hormone substitutes, and hormone antagonists - Abstract
The design and synthesis of 4-hydroxytamoxifen (4-OHT) derivatives are described. The binding affinities of these compounds toward the orphan estrogen-related receptor gamma and the classical estrogen receptor alpha demonstrate that analogs bearing hydroxyalkyl groups display improved binding selectivity profiles compared with that of 4-OHT. An X-ray crystal structure of one of the designed compounds bound to ERRgamma LBD confirms the molecular basis of the selectivity.
- Published
- 2006
17. Discovery of a novel and potent series of dianilinopyrimidineurea and urea isostere inhibitors of VEGFR2 tyrosine kinase
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Anne T. Truesdale, Liping Wang, Jeffrey A. Stafford, James Marvin Veal, Robert T. Nolte, Victoria B. Knick, Sharon K. Rudolph, Mui Cheung, Kristen Elizabeth Nailor, Eldridge N. Nartey, Douglas M. Sammond, and Rakesh Kumar
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Isostere ,Clinical Biochemistry ,Pharmaceutical Science ,Biochemistry ,Structure-Activity Relationship ,Drug Discovery ,Urea ,Structure–activity relationship ,Potency ,Enzyme Inhibitors ,Molecular Biology ,Aniline Compounds ,biology ,Chemistry ,Kinase ,Organic Chemistry ,Stereoisomerism ,Kinase insert domain receptor ,HIV Protease Inhibitors ,Protein-Tyrosine Kinases ,respiratory system ,Vascular Endothelial Growth Factor Receptor-2 ,Pyrimidines ,Enzyme inhibitor ,cardiovascular system ,biology.protein ,Molecular Medicine ,Signal transduction ,Tyrosine kinase ,circulatory and respiratory physiology - Abstract
A series of dianilinopyrimidineureas demonstrate potency as VEGFR2 kinase inhibitors.
- Published
- 2005
18. Discovery and Evaluation of 2-Anilino-5-aryloxazoles as a Novel Class of VEGFR2 Kinase Inhibitors
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Wendy Liu, James Marvin Veal, Deirdre K. Luttrell, Mui Cheung, Jeffrey A. Stafford, Robert T. Nolte, Robert N. Hunter, Jennifer H. Johnson, Renae M. Crosby, Liping Wang, Matthew L. Brown, Philip A. Harris, Rakesh Kumar, and Andrea H. Epperly
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Male ,Models, Molecular ,Vascular Endothelial Growth Factor A ,Umbilical Veins ,Stereochemistry ,Angiogenesis Inhibitors ,Crystallography, X-Ray ,Ligands ,Sulfone ,Rats, Sprague-Dawley ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Meta ,Adenosine Triphosphate ,Dogs ,In vivo ,Drug Discovery ,Animals ,Humans ,Structure–activity relationship ,Oxazoles ,Cells, Cultured ,Cell Proliferation ,Oxazole ,chemistry.chemical_classification ,Aniline Compounds ,Binding Sites ,biology ,Aryl ,Aromatic amine ,Vascular Endothelial Growth Factor Receptor-2 ,Xenograft Model Antitumor Assays ,Rats ,Solubility ,chemistry ,Enzyme inhibitor ,biology.protein ,Molecular Medicine - Abstract
A series of derivatives of 2-anilino-5-phenyloxazole (5) has been identified as inhibitors of VEGFR2 kinase. Herein we describe the structure-activity relationship (SAR) of this novel template. Optimization of both aryl rings led to very potent inhibitors at both the enzymatic and cellular levels. Oxazole 39 had excellent solubility and good oral PK when dosed as the bis-mesylate salt and demonstrated moderate in vivo efficacy against HT29 human colon tumor xenografts. X-ray crystallography confirmed the proposed binding mode, and comparison of oxazoles 39 and 46 revealed interesting differences in orientation of 2-pyridyl and 3-pyridyl rings, respectively, attached at the meta position of the 5-phenyl ring.
- Published
- 2005
19. The Crystal Structure of Nucleoplasmin-Core
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Kari L. Hartman, Shuchismita Dutta, Christopher W. Akey, Ildikó V. Akey, Tom Laue, James F. Head, Colin Dingwall, and Robert T. Nolte
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Genetics ,Nucleoplasmin ,education.field_of_study ,biology ,Nucleosome assembly ,Pentamer ,Cell Biology ,Histone ,Sperm chromatin decondensation ,Chaperone (protein) ,biology.protein ,Biophysics ,Nucleosome ,education ,Molecular Biology ,Histone binding - Abstract
The efficient assembly of histone complexes and nucleosomes requires the participation of molecular chaperones. Currently, there is a paucity of data on their mechanism of action. We now present the structure of an N-terminal domain of nucleoplasmin (Np-core) at 2.3 A resolution. The Np-core monomer is an eight-stranded β barrel that fits snugly within a stable pentamer. In the crystal, two pentamers associate to form a decamer. We show that both Np and Np-core are competent to assemble large complexes that contain the four core histones. Further experiments and modeling suggest that these complexes each contain five histone octamers which dock to a central Np decamer. This work has important ramifications for models of histone storage, sperm chromatin decondensation, and nucleosome assembly.
- Published
- 2001
20. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation
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David W. Rose, Nuria Assa-Munt, Joseph Torchia, Michael V. Milburn, Tina-Marie Mullen, Stefan Westin, Anna Krones, Michael G. Rosenfeld, Robert T. Nolte, Eileen M. McInerney, Juan Inostroza, Sarah E. Flynn, and Christopher K. Glass
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Models, Molecular ,Transcriptional Activation ,Receptors, Steroid ,Molecular Sequence Data ,Receptors, Cytoplasmic and Nuclear ,Estrogen receptor ,Biology ,Protein Structure, Secondary ,Nuclear Receptor Coactivator 1 ,Coactivator ,Genetics ,Consensus sequence ,Animals ,Amino Acid Sequence ,Transcription factor ,Cells, Cultured ,Histone Acetyltransferases ,Nuclear Proteins ,Fibroblasts ,Peptide Fragments ,Rats ,Cell biology ,Nuclear receptor coactivator 1 ,Gene Expression Regulation ,Nuclear receptor ,Biochemistry ,Nuclear receptor coactivator 3 ,Trans-Activators ,Nuclear receptor coactivator 2 ,Sequence Alignment ,Research Paper ,Transcription Factors ,Developmental Biology - Abstract
Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3′) in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARγ activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.
- Published
- 1998
21. Interactions controlling the assembly of nuclear-receptor heterodimers and co-activators
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D W Rose, Stefan Westin, Wisely Gb, Michael V. Milburn, Eileen M. McInerney, Michael G. Rosenfeld, Robert T. Nolte, Riki Kurokawa, and Christopher K. Glass
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Models, Molecular ,Conformational change ,Protein Conformation ,Receptors, Retinoic Acid ,Stereochemistry ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Allosteric regulation ,Receptors, Cytoplasmic and Nuclear ,Peroxisome proliferator-activated receptor ,Retinoid X receptor ,Biology ,Ligands ,Benzoates ,DNA-binding protein ,Cell Line ,Retinoids ,Nuclear Receptor Coactivator 1 ,Allosteric Regulation ,Amino Acid Sequence ,Receptor ,Transcription factor ,Histone Acetyltransferases ,chemistry.chemical_classification ,Binding Sites ,Multidisciplinary ,organic chemicals ,Cell biology ,body regions ,Retinoid X Receptors ,Nuclear receptor ,chemistry ,embryonic structures ,lipids (amino acids, peptides, and proteins) ,Transcription Factors - Abstract
Retinoic-acid receptor-α (RAR-α) and peroxisome proliferator-activated receptor-γ (PPAR-γ) are members of the nuclear-receptor superfamily that bind to DNA as heterodimers with retinoid-X receptors (RXRs)1,2. PPAR–RXR heterodimers can be activated by PPAR or RXR ligands3, whereas RAR–RXR heterodimers are selectively activated by RAR ligands only, because of allosteric inhibition of the binding of ligands to RXR by RAR4,5. However, RXR ligands can potentiate the transcriptional effects of RAR ligands in cells6. Transcriptional activation by nuclear receptors requires a carboxy-terminal helical region, termed activation function-2 (AF-2) (refs 7,8,9), that forms part of the ligand-binding pocket and undergoes a conformational change required for the recruitment of co-activator proteins, including NCoA-1/SRC-1 (refs 10,11,12,13,14,15,16,17). Here we show that allosteric inhibition of RXR results from a rotation of the RXR AF-2 helix that places it in contact with the RAR coactivator-binding site. Recruitment of an LXXLL motif of SRC-1 to RAR in response to ligand displaces the RXR AF-2 domain, allowing RXR ligands to bind and promote the binding of a second LXXLL motif from the same SRC-1 molecule. These results may partly explain the different responses of nuclear-receptor heterodimers to RXR-specific ligands.
- Published
- 1998
22. Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis
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Kirsten M. Kahler, Philip A. Harris, Paris Ward, Michael T. Ouellette, Sandra J. Hoffman, Lauren Dare, Peter J. Gough, Helen H. Sun, Carol A. Capriotti, Michelle C. Schaeffer, Christina S. Pao, Robert T. Nolte, Joshua N. Finger, Nino Campobasso, Ruth Lehr, Angela Smallwood, Deepak Bandyopadhyay, Rachel D. Totoritis, Robert W. Marquis, John D. Lich, Rakesh Nagilla, Scott B. Berger, Barbara A. Swift, John Bertin, and Julie A. Cox
- Subjects
Pyrimidine ,Kinase ,business.industry ,Necroptosis ,Organic Chemistry ,Pharmacology ,Hypothermia ,Biochemistry ,Small molecule ,chemistry.chemical_compound ,chemistry ,Drug Discovery ,medicine ,Transferase ,Tumor necrosis factor alpha ,medicine.symptom ,business - Abstract
Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.
- Published
- 2013
23. Dolutegravir interactions with HIV-1 integrase-DNA: structural rationale for drug resistance and dissociation kinetics
- Author
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Mark R. Underwood, Felix Deanda, Kazunari Hattori, Tomokazu Yoshinaga, Kendra E. Hightower, Robert T. Nolte, and Takashi Kawasuji
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Models, Molecular ,Protein Conformation ,Pyridones ,Mutant ,Integrase inhibitor ,lcsh:Medicine ,Drug resistance ,HIV Integrase ,Pharmacology ,Piperazines ,chemistry.chemical_compound ,Proviruses ,Drug Resistance, Viral ,Oxazines ,medicine ,Humans ,HIV Integrase Inhibitors ,lcsh:Science ,HIV Long Terminal Repeat ,Multidisciplinary ,biology ,Molecular Structure ,Elvitegravir ,lcsh:R ,Raltegravir ,Virology ,Integrase ,Molecular Docking Simulation ,Kinetics ,chemistry ,Dolutegravir ,biology.protein ,HIV-1 ,Nucleic Acid Conformation ,lcsh:Q ,Heterocyclic Compounds, 3-Ring ,DNA ,medicine.drug ,Protein Binding ,Research Article - Abstract
Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.
- Published
- 2013
24. Structure of the IRS-1 PTB Domain Bound to the Juxtamembrane Region of the Insulin Receptor
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Michael J. Eck, Steven E. Shoelson, Sirano Dhe-Paganon, Thomas Trüb, and Robert T. Nolte
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Phosphotyrosine binding ,Models, Molecular ,Insulin Receptor Substrate Proteins ,Protein Conformation ,Molecular Sequence Data ,Crystallography, X-Ray ,Peptide Mapping ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,src Homology Domains ,Protein structure ,Insulin receptor substrate ,Humans ,Amino Acid Sequence ,Phosphotyrosine ,Binding Sites ,biology ,Molecular Structure ,Biochemistry, Genetics and Molecular Biology(all) ,Phosphoproteins ,Receptor, Insulin ,IRS1 ,Cell biology ,Pleckstrin homology domain ,Insulin receptor ,Biochemistry ,biology.protein ,Phosphotyrosine-binding domain - Abstract
Crystal structures of the insulin receptor substrate-1 (IRS-1) phosphotyrosine-binding (PTB) domain, alone and complexed with the juxtamembrane region of the insulin receptor, show how this domain recognizes phosphorylated "NPXY" sequence motifs. The domain is a 7-stranded β sandwich capped by a C-terminal helix. The insulin receptor phosphopeptide fills an L-shaped cleft on the domain. The N-terminal residues of the bound peptide form an additional strand in the β sandwich, stabilized by contacts with the C-terminal helix. These interactions explain why IRS-1 binds to the insulin receptor but not to NPXpY motifs in growth factor receptors. The PTB domains of IRS-1 and Shc share a common fold with pleckstrin homology domains. Overall, ligand binding by IRS-1 and Shc PTB domains is similar, but residues critical for phosphotyrosine recognition are not conserved.
- Published
- 1996
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25. Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization, core and urea optimization and in vivo efficacy
- Author
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David M. Bickett, Pierette Banker, Daphne C. Clancy, Joyce A. Boucheron, Stephen A. Thomson, James E. Weiel, and Andrew J. Peat, Dulce Garrido, Steven M. Sparks, Francis X. Tavares, Lauren R. Sheckler, Joel P. Cooper, Scott Howard Dickerson, Liping Wang, Tony Y. Wang, Robert T. Nolte, and H.L. Carter
- Subjects
Blood Glucose ,Clinical Biochemistry ,Molecular Conformation ,Pharmaceutical Science ,Crystallography, X-Ray ,Biochemistry ,Glucagon ,Glycogen phosphorylase ,chemistry.chemical_compound ,Mice ,Structure-Activity Relationship ,In vivo ,Drug Discovery ,Transferase ,Potency ,Structure–activity relationship ,Animals ,Combinatorial Chemistry Techniques ,Hypoglycemic Agents ,Urea ,ortho-Aminobenzoates ,Molecular Biology ,Glycogen ,Molecular Structure ,Chemistry ,Organic Chemistry ,Glycogen Phosphorylase ,Crystallography ,Disease Models, Animal ,Diabetes Mellitus, Type 2 ,Molecular Medicine - Abstract
Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.
- Published
- 2008
26. Discovery of 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide (Pazopanib), a novel and potent vascular endothelial growth factor receptor inhibitor
- Author
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Jeffrey A. Stafford, Jennifer H. Johnson, Philip A. Harris, Robert A. Mook, James Marvin Veal, Christopher P. Laudeman, Ronda G. Davis-Ward, Mui Cheung, Szewczyk Jerzy Ryszard, Robert N. Hunter, Kevin Hinkle, Deirdre K. Luttrell, Amogh Boloor, Liping Wang, Robert T. Nolte, Anne T. Truesdale, Sharon K. Rudolph, Renae M. Crosby, Rakesh Kumar, Victoria B. Knick, and Andrea H. Epperly
- Subjects
Models, Molecular ,Indazoles ,Crystallography, X-Ray ,Pazopanib ,chemistry.chemical_compound ,Mice ,Growth factor receptor ,Cytochrome P-450 Enzyme System ,In vivo ,Neoplasms ,Drug Discovery ,medicine ,Animals ,Cytochrome P-450 Enzyme Inhibitors ,Humans ,Receptor ,Protein Kinase Inhibitors ,Cells, Cultured ,Sulfonamides ,Molecular Structure ,Cancer ,medicine.disease ,Xenograft Model Antitumor Assays ,Vascular endothelial growth factor ,Endothelial stem cell ,Isoenzymes ,Pyrimidines ,Receptors, Vascular Endothelial Growth Factor ,Biochemistry ,chemistry ,Cancer research ,Molecular Medicine ,Signal transduction ,medicine.drug - Abstract
Inhibition of the vascular endothelial growth factor (VEGF) signaling pathway has emerged as one of the most promising new approaches for cancer therapy. We describe herein the key steps starting from an initial screening hit leading to the discovery of pazopanib, N(4)-(2,3-dimethyl-2H-indazol-6-yl)-N(4)-methyl-N(2)-(4-methyl-3-sulfonamidophenyl)-2,4-pyrimidinediamine, a potent pan-VEGF receptor (VEGFR) inhibitor under clinical development for renal-cell cancer and other solid tumors.
- Published
- 2008
27. X-ray crystal structures of the estrogen-related receptor-gamma ligand binding domain in three functional states reveal the molecular basis of small molecule regulation
- Author
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Thomas G. Consler, William J. Zuercher, David D. McKee, Lisa A. Orband-Miller, Aaron B. Miller, Timothy M. Willson, Liping Wang, Millard H. Lambert, and Robert T. Nolte
- Subjects
Agonist ,Models, Molecular ,Stereochemistry ,medicine.drug_class ,Static Electricity ,Receptors, Cytoplasmic and Nuclear ,Peptide ,Peptide binding ,In Vitro Techniques ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Estrogen-related receptor ,medicine ,Inverse agonist ,Humans ,Receptor ,Protein Structure, Quaternary ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,Chemistry ,Circular Dichroism ,Cell Biology ,Small molecule ,Recombinant Proteins ,Protein Structure, Tertiary ,Nuclear receptor ,Receptors, Estrogen ,Multiprotein Complexes ,hormones, hormone substitutes, and hormone antagonists - Abstract
X-ray crystal structures of the ligand binding domain (LBD) of the estrogen-related receptor-gamma (ERRgamma) were determined that describe this receptor in three distinct states: unliganded, inverse agonist bound, and agonist bound. Two structures were solved for the unliganded state, the ERRgamma LBD alone, and in complex with a coregulator peptide representing a portion of receptor interacting protein 140 (RIP140). No significant differences were seen between these structures that both exhibited the conformation of ERRgamma seen in studies with other coactivators. Two structures were obtained describing the inverse agonist-bound state, the ERRgamma LBD with 4-hydroxytamoxifen (4-OHT), and the ERRgamma LBD with 4-OHT and a peptide representing a portion of the silencing mediator of retinoid and thyroid hormone action protein (SMRT). The 4-OHT structure was similar to other reported inverse agonist bound structures, showing reorientation of phenylalanine 435 and a displacement of the AF-2 helix relative to the unliganded structures with little other rearrangement occurring. No significant changes to the LBD appear to be induced by peptide binding with the addition of the SMRT peptide to the ERRgamma plus 4-OHT complex. The observed agonist-bound state contains the ERRgamma LBD, a ligand (GSK4716), and the RIP140 peptide and reveals an unexpected rearrangement of the phenol-binding residues. Thermal stability studies show that agonist binding leads to global stabilization of the ligand binding domain. In contrast to the conventional mechanism of nuclear receptor ligand activation, activation of ERRgamma by GSK4716 does not appear to involve a major rearrangement or significant stabilization of the C-terminal helix.
- Published
- 2006
28. Structure-Guided Synthesis of Tamoxifen Analogues with Improved Selectivity for the Orphan ERRγ
- Author
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Lisa A. Orband-Miller, Jon L. Collins, Donald P. McDonnell, Timothy M. Willson, Esther Y. Chao, Liping Wang, Robert T. Nolte, Stephanie Gaillard, Aaron B. Miller, and William J. Zuercher
- Subjects
Chemistry ,medicine ,General Medicine ,Selectivity ,Combinatorial chemistry ,Tamoxifen ,medicine.drug - Published
- 2006
29. Crystallization of protein-ligand complexes
- Author
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Robert X. Xu, Lisa M. Shewchuk, Robert T. Gampe, Tamara E. Grisard, Shawn P. Williams, Gang An, Liping Wang, Su-Jun J. Deng, Warren J. Rocque, Anne M. Hassell, Robert T. Nolte, Kurt Weaver, Kevin P. Madauss, H. Luke Carter, Jane Bynum, G. Bruce Wisely, and Randy K. Bledsoe
- Subjects
crystallization ,Stereochemistry ,Ab initio ,Molecular Conformation ,Crystallography, X-Ray ,Ligands ,Cofactor ,Receptors, Glucocorticoid ,Structural Biology ,Protein purification ,Molecule ,Animals ,Humans ,Binding site ,Binding Sites ,biology ,Chemistry ,Temperature ,Proteins ,General Medicine ,Small molecule ,Research Papers ,Receptors, Mineralocorticoid ,Receptors, Androgen ,Liposomes ,Mutation ,biology.protein ,Nucleic acid ,protein–ligand complexes ,Carrier Proteins ,Protein ligand - Abstract
Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals., Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein–ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.
- Published
- 2006
30. Crystal structures of the catalytic domain of phosphodiesterase 4B complexed with AMP, 8-Br-AMP, and rolipram
- Author
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Michael A. Luther, Robert X. Xu, Dana E. Vanderwall, Warren J. Rocque, Robert T. Nolte, and Millard H. Lambert
- Subjects
Models, Molecular ,Stereochemistry ,Macromolecular Substances ,Phosphodiesterase Inhibitors ,Molecular Sequence Data ,Static Electricity ,In Vitro Techniques ,Crystallography, X-Ray ,PDE4B ,Adenosine Triphosphate ,Structural Biology ,Catalytic Domain ,Hydrolase ,medicine ,Humans ,Nucleotide ,Amino Acid Sequence ,Molecular Biology ,Rolipram ,chemistry.chemical_classification ,Binding Sites ,biology ,Sequence Homology, Amino Acid ,Hydrogen bond ,Phosphodiesterase ,Active site ,Adenosine Monophosphate ,Cyclic Nucleotide Phosphodiesterases, Type 4 ,Protein Structure, Tertiary ,Enzyme ,chemistry ,3',5'-Cyclic-AMP Phosphodiesterases ,Mutation ,biology.protein ,medicine.drug - Abstract
Phosphodiesterase catalyzes the hydrolysis of the intracellular second messenger 3′,5′-cyclic AMP (cAMP) into the corresponding 5′-nucleotide. Phosphodiesterase 4 (PDE4), the major cAMP-specific PDE in inflammatory and immune cells, is an attractive target for the treatment of asthma and COPD. We have determined crystal structures of the catalytic domain of PDE4B complexed with AMP (2.0 A), 8-Br-AMP (2.13 A) and the potent inhibitor rolipram (2.0 A). All the ligands bind in the same hydrophobic pocket and can interact directly with the active site metal ions. The identity of these metal ions was examined using X-ray anomalous difference data. The structure of the AMP complex confirms the location of the catalytic site and allowed us to speculate about the detailed mechanism of catalysis. The high-resolution structures provided the experimental insight into the nucleotide selectivity of phosphodiesterase. 8-Br-AMP binds in the syn conformation to the enzyme and demonstrates an alternative nucleotide-binding mode. Rolipram occupies much of the AMP-binding site and forms two hydrogen bonds with Gln443 similar to the nucleotides.
- Published
- 2003
31. Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARalpha
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Millard H. Lambert, Kelli D. Plunket, Jeffery E. Cobb, John T. Moore, Cristin M. Galardi, Derek J. Parks, H. Eric Xu, D.D. McKee, Robert T. Nolte, Julie B. Stimmel, Timothy M. Willson, Valerie G. Montana, Steven A. Kliewer, Barry G. Shearer, and Thomas B. Stanley
- Subjects
Models, Molecular ,Amino Acid Motifs ,Molecular Sequence Data ,Receptors, Cytoplasmic and Nuclear ,Biology ,Crystallography, X-Ray ,Ligands ,Protein Structure, Secondary ,Inhibitory Concentration 50 ,Structure-Activity Relationship ,Humans ,Nuclear Receptor Co-Repressor 2 ,Amino Acid Sequence ,Receptor ,Oxazoles ,Nuclear receptor co-repressor 1 ,PELP-1 ,Nuclear receptor co-repressor 2 ,Multidisciplinary ,Binding Sites ,Cell biology ,Protein Structure, Tertiary ,Nuclear receptor coactivator 1 ,DNA-Binding Proteins ,Repressor Proteins ,Nuclear receptor ,Thyroid hormone receptor alpha ,Biochemistry ,Small heterodimer partner ,Tyrosine ,Sequence Alignment ,Protein Binding ,Transcription Factors - Abstract
Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N-CoR1,2, which in turn recruit histone deacetylases to the chromatin3,4,5. Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome6,7,8. The binding of co-repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists9. Here we report the crystal structure of a ternary complex containing the peroxisome proliferator-activated receptor-α ligand-binding domain bound to the antagonist GW6471 and a SMRT co-repressor motif. In this structure, the co-repressor motif adopts a three-turn α-helix that prevents the carboxy-terminal activation helix (AF-2) of the receptor from assuming the active conformation. Binding of the co-repressor motif is further reinforced by the antagonist, which blocks the AF-2 helix from adopting the active position. Biochemical analyses and structure-based mutagenesis indicate that this mode of co-repressor binding is highly conserved across nuclear receptors.
- Published
- 2002
32. Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity
- Author
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Michael A. Luther, Michael V. Milburn, Hengming Ke, Dana E. Vanderwall, Millard H. Lambert, Warren J. Rocque, Robert X. Xu, Robert T. Nolte, Yingdong Zhao, William D. Holmes, and Anne M. Hassell
- Subjects
Models, Molecular ,Protein Folding ,Protein Conformation ,Neurotransmission ,Crystallography, X-Ray ,Exocytosis ,Protein Structure, Secondary ,Substrate Specificity ,Catalytic Domain ,medicine ,Cyclic AMP ,Nucleotide ,Cyclic GMP ,chemistry.chemical_classification ,Multidisciplinary ,Binding Sites ,biology ,Hydrolysis ,Phosphodiesterase ,Active site ,Hydrogen Bonding ,Cyclic Nucleotide Phosphodiesterases, Type 4 ,Biochemistry ,chemistry ,Mechanism of action ,3',5'-Cyclic-AMP Phosphodiesterases ,Metals ,Second messenger system ,biology.protein ,medicine.symptom ,Crystallization ,Intracellular - Abstract
Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.
- Published
- 2000
33. Crystal structure of the pleckstrin homology-phosphotyrosine binding (PH-PTB) targeting region of insulin receptor substrate 1
- Author
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Michael J. Eck, Sirano Dhe-Paganon, Steven E. Shoelson, Elizabeth A. Ottinger, and Robert T. Nolte
- Subjects
Phosphotyrosine binding ,Models, Molecular ,Phosphatidylinositol 4,5-Diphosphate ,Insulin Receptor Substrate Proteins ,Molecular Sequence Data ,Peptide binding ,Crystallography, X-Ray ,Protein Structure, Secondary ,Humans ,Amino Acid Sequence ,Binding site ,Phosphotyrosine ,Multidisciplinary ,Binding Sites ,Chemistry ,Biological Sciences ,Phosphoproteins ,IRS1 ,Pleckstrin homology domain ,Crystallography ,Biophysics ,Phosphotyrosine-binding domain ,Sequence Alignment ,Binding domain ,Protein Binding ,Signal Transduction - Abstract
We have determined the crystal structure at 2.3-Å resolution of an amino-terminal segment of human insulin receptor substrate 1 that encompasses its pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Both domains adopt the canonical seven-stranded β-sandwich PH domain fold. The domains are closely associated, with a 720-Å 2 contact surface buried between them that appears to be stabilized by ionic, hydrophobic, and hydrogen bonding interactions. The nonconserved 46-residue linker between the domains is disordered. The PTB domain peptide binding site is fully exposed on the molecular surface, as is a large cationic patch at the base of the PH domain that is a likely binding site for the head groups of phosphatidylinositol phosphates. Binding assays confirm that phosphatidylinositol phosphates bind the PH domain, but not the PTB domain. Ligand binding to the PH domain does not alter PTB domain interactions, and vice versa. The structural and accompanying functional data illustrate how the two binding domains might act cooperatively to effectively increase local insulin receptor substrate 1 concentration at the membrane and transiently fix the receptor and substrate, to allow multiple phosphorylation reactions to occur during each union.
- Published
- 1999
34. Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma
- Author
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Stefan Westin, Timothy M. Willson, Riki Kurokawa, G. B. Wisely, Michael G. Rosenfeld, Cobb Jeffrey Edmond, Robert T. Nolte, Christopher K. Glass, Michael V. Milburn, and Millard H. Lambert
- Subjects
Transcriptional Activation ,Stereochemistry ,Macromolecular Substances ,Protein Conformation ,Molecular Sequence Data ,Peroxisome proliferator-activated receptor ,Receptors, Cytoplasmic and Nuclear ,Biology ,Crystallography, X-Ray ,Ligands ,Rosiglitazone ,Structure-Activity Relationship ,Protein structure ,Nuclear Receptor Coactivator 1 ,Escherichia coli ,Glucose homeostasis ,Humans ,Hypoglycemic Agents ,Amino Acid Sequence ,Binding site ,Cloning, Molecular ,Conserved Sequence ,Histone Acetyltransferases ,chemistry.chemical_classification ,Multidisciplinary ,Binding Sites ,Ligand (biochemistry) ,Protein Structure, Tertiary ,Nuclear receptor coactivator 1 ,Thiazoles ,chemistry ,Nuclear receptor ,Nuclear receptor coactivator 3 ,Thiazolidinediones ,Transcription Factors - Abstract
The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma. We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.
- Published
- 1998
35. Differing roles for zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex
- Author
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Raymond S. Brown, Robert T. Nolte, Rachel M. Conlin, and Stephen C. Harrison
- Subjects
Models, Molecular ,Protein Conformation ,Molecular Sequence Data ,Xenopus ,Crystallography, X-Ray ,DNA-binding protein ,DNA, Ribosomal ,chemistry.chemical_compound ,5S ribosomal RNA ,Xenopus laevis ,Transcription Factor TFIIIA ,Animals ,Amino Acid Sequence ,Promoter Regions, Genetic ,Transcription factor ,Zinc finger ,Multidisciplinary ,biology ,Base Sequence ,RNA, Ribosomal, 5S ,Promoter ,Zinc Fingers ,Biological Sciences ,biology.organism_classification ,Molecular biology ,Peptide Fragments ,Cell biology ,DNA-Binding Proteins ,chemistry ,Nucleic Acid Conformation ,DNA ,Protein Binding ,Transcription Factors - Abstract
The crystal structure of the six NH 2 -terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound with 31 bp of the 5S rRNA gene promoter has been determined at 3.1 Å resolution. Individual zinc fingers are positioned differently in the major groove and across the minor groove of DNA to span the entire length of the duplex. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove.
- Published
- 1998
36. Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes
- Author
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Steven E. Shoelson, Stephen C. Harrison, Joseph Schlessinger, Michael J. Eck, and Robert T. Nolte
- Subjects
Models, Molecular ,Phosphopeptides ,Stereochemistry ,Protein Conformation ,Protein subunit ,Molecular Sequence Data ,Peptide ,Peptide binding ,SH2 domain ,Crystallography, X-Ray ,src Homology Domains ,chemistry.chemical_compound ,Phosphatidylinositol 3-Kinases ,Methionine ,Structural Biology ,Humans ,Receptors, Platelet-Derived Growth Factor ,Phosphatidylinositol ,Amino Acid Sequence ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,Phosphopeptide ,Phosphotransferases (Alcohol Group Acceptor) ,Proto-Oncogene Proteins c-kit ,chemistry ,Biochemistry ,Phosphoprotein ,Proto-oncogene tyrosine-protein kinase Src ,Protein Binding - Abstract
Crystal structures of the amino-terminal SH2 domain of the p85alpha subunit of phosphatidylinositol (PI) 3-kinase, alone and in complex with phosphopeptides bearing pTyr-Met/Val-Xaa-Met motifs, show that phosphopeptides bind in the two-pronged manner seen in high-affinity Lck and Src SH2 complexes, with conserved interactions between the domain and the peptide segment from phosphotyrosine to Met+3. Peptide binding requires the rearrangement of a tyrosyl side chain in the BG loop to create the hydrophobic Met+3 binding pocket. The structures suggest a mechanism for the biological specificity exhibited by PI 3-kinase in its interactions with phosphoprotein partners.
- Published
- 1996
37. Discovery of 5-[[4-[(2,3-Dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide (Pazopanib), a Novel and Potent Vascular Endothelial Growth Factor Receptor Inhibitor.
- Author
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Philip A. Harris, Amogh Boloor, Mui Cheung, Rakesh Kumar, Renae M. Crosby, Ronda G. Davis-Ward, Andrea H. Epperly, Kevin W. Hinkle, Robert N. Hunter III, Jennifer H. Johnson, Victoria B. Knick, Christopher P. Laudeman, Deirdre K. Luttrell, Robert A. Mook, Robert T. Nolte, Sharon K. Rudolph, Jerzy R. Szewczyk, Anne T. Truesdale, James M. Veal, and Liping Wang
- Published
- 2008
- Full Text
- View/download PDF
38. Discovery of Novel Benzimidazoles as Potent Inhibitors of TIE-2 and VEGFR-2 Tyrosine Kinase Receptors.
- Author
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Masaichi Hasegawa, Naohiko Nishigaki, Yoshiaki Washio, Kazuya Kano, Philip A. Harris, Hideyuki Sato, Ichiro Mori, Rob I. West, Megumi Shibahara, Hiroko Toyoda, Liping Wang, Robert T. Nolte, James M. Veal, and Mui Cheung
- Published
- 2007
- Full Text
- View/download PDF
39. The amino blocking reagent 1-isopropyl-3-ethoxy-4-nitro-2-oxo-3-pyrroline and the N-hydroxysuccinimide esters of N-(1-cyclohexyl- and 1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)glycine. Reagents for the introduction of N-glycyl residues
- Author
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Mark A. Koshute, James R. Miller, William E. Brown, George K. Chin, Kimberly E. Niemela, Philip L. Southwick, Cheryl A. Siegel, and Robert T. Nolte
- Subjects
chemistry.chemical_compound ,Chemistry ,Reagent ,Organic Chemistry ,Alkoxy group ,Nitro ,Lactam ,Tripeptide ,Pyrroline ,Protecting group ,Medicinal chemistry ,Isopropyl - Published
- 1984
40. The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms
- Author
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Margarita Hadzopoulou-Cladaras, Vassilis I. Zannis, C Cladaras, David Atkinson, and Robert T. Nolte
- Subjects
Untranslated region ,Adult ,Apolipoprotein B ,Transcription, Genetic ,Sequence analysis ,General Biochemistry, Genetics and Molecular Biology ,Protein sequencing ,Sequence Homology, Nucleic Acid ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,Protein secondary structure ,Peptide sequence ,Apolipoproteins B ,chemistry.chemical_classification ,General Immunology and Microbiology ,biology ,Base Sequence ,General Neuroscience ,Protein primary structure ,DNA ,DNA Restriction Enzymes ,Molecular biology ,Amino acid ,Biochemistry ,chemistry ,Genes ,Liver ,Receptors, LDL ,Apolipoprotein B-100 ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Apolipoprotein B-48 ,Research Article - Abstract
We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B-100 (apoB-100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5' and 3' untranslated regions which are 128 and 301 nucleotides respectively. The DNA-derived protein sequence shows that apoB-100 is 513,000 daltons and contains 4560 amino acids including a 24-amino-acid-long signal peptide. The mol. wt of apoB-100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non-amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid-binding domains of apoB-100. The protein contains 25 cysteines and 20 potential N-glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor-binding domain of apoB-100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15-kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB-100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.
- Published
- 1986
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