19 results on '"May EW"'
Search Results
2. Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.
- Author
-
Wittlinger F, Ogboo BC, Shevchenko E, Damghani T, Pham CD, Schaeffner IK, Oligny BT, Chitnis SP, Beyett TS, Rasch A, Buckley B, Urul DA, Shaurova T, May EW, Schaefer EM, Eck MJ, Hershberger PA, Poso A, Laufer SA, and Heppner DE
- Abstract
Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
3. Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors.
- Author
-
Hoyt KW, Urul DA, Ogboo BC, Wittlinger F, Laufer SA, Schaefer EM, May EW, and Heppner DE
- Subjects
- Reproducibility of Results, Drug Design, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors chemistry, ErbB Receptors, Enzyme Inhibitors pharmacology
- Abstract
Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.
- Published
- 2024
- Full Text
- View/download PDF
4. Structural Basis for Inhibition of Mutant EGFR with Lazertinib (YH25448).
- Author
-
Heppner DE, Wittlinger F, Beyett TS, Shaurova T, Urul DA, Buckley B, Pham CD, Schaeffner IK, Yang B, Ogboo BC, May EW, Schaefer EM, Eck MJ, Laufer SA, and Hershberger PA
- Abstract
Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)
- Published
- 2022
- Full Text
- View/download PDF
5. Erratum to: PGRMC1 contributes to doxorubicin-induced chemoresistance in MES-SA uterine sarcoma.
- Author
-
Lin ST, May EW, Chang JF, Hu RY, Wang LH, and Chan HL
- Published
- 2015
- Full Text
- View/download PDF
6. PGRMC1 contributes to doxorubicin-induced chemoresistance in MES-SA uterine sarcoma.
- Author
-
Lin ST, May EW, Chang JF, Hu RY, Wang LH, and Chan HL
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Apoptosis, Blotting, Western, Cell Adhesion, Cell Cycle, Cell Movement, Cell Proliferation, Female, Flow Cytometry, Fluorescent Antibody Technique, Humans, Membrane Proteins antagonists & inhibitors, Membrane Proteins genetics, RNA, Small Interfering genetics, Receptors, Progesterone antagonists & inhibitors, Receptors, Progesterone genetics, Sarcoma genetics, Sarcoma pathology, Signal Transduction, Tumor Cells, Cultured, Uterine Neoplasms genetics, Uterine Neoplasms pathology, Antibiotics, Antineoplastic pharmacology, Doxorubicin pharmacology, Drug Resistance, Neoplasm, Epithelial-Mesenchymal Transition, Membrane Proteins metabolism, Receptors, Progesterone metabolism, Sarcoma drug therapy, Uterine Neoplasms drug therapy
- Abstract
Chemotherapy is one of the major categories of medical oncology and a primary tumor treatment; however, the effectiveness of chemotherapy is restricted by drug resistance. Overcoming resistance to chemotherapy and investigating molecular targeted therapies are challenges currently faced during resistance management. Progesterone receptor membrane component 1 (PGRMC1) is an adapter protein mediating cholesterol synthesis, steroid signaling, and cytochrome p450 activation. Attention has recently focused on the role of PGRMC1 in cell survival, anti-apoptosis, and damage response. In the present study, we used knockdown and overexpression approaches in the following set of uterine sarcoma models to further evaluate the role of PGRMC1 in drug resistance: the doxorubicin-sensitive MES-SA cells and the doxorubicin-resistant MES-SA/DxR-2 µM and MES-SA/DxR-8 µM cells (with different levels of doxorubicin resistance). PGRMC1 repressed doxorubicin-induced cytotoxicity and exhibited an anti-apoptotic effect; it also promoted cell proliferation and cell cycle progression to the S phase. Of note, PGRMC1 overexpression led to the epithelial-mesenchymal transition (EMT) of the sensitive MES-SA cells, thus facilitating their migration and invasion. The combination of PGRMC1 knockdown and the P-glycoprotein inhibitor verapamil significantly decreased the viability of P-glycoprotein-overexpressing MES-SA/DxR-8 μM cells after doxorubicin treatment. Taken together, our results show that PGRMC1 contributed to chemoresistance through cell proliferation, anti-apoptosis, and EMT induction, leading to the suggestion that PGRMC1 may serve as a therapeutic target in combination with an inhibitor in different drug resistance pathways and indicating the usefulness of predictive resistance biomarkers in uterine sarcoma.
- Published
- 2015
- Full Text
- View/download PDF
7. Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.
- Author
-
Wanner J, Romashko D, Werner DS, May EW, Peng Y, Schulz R, Foreman KW, Russo S, Arnold LD, Pingle M, Bergstrom DE, Barany F, and Thomson S
- Subjects
- Basic Helix-Loop-Helix Leucine Zipper Transcription Factors biosynthesis, Cell Line, Tumor, Drug Design, Gene Expression Regulation, Neoplastic drug effects, Glycols chemistry, Humans, Ligands, Neoplasms drug therapy, Protein Interaction Maps drug effects, Proto-Oncogene Proteins c-myc antagonists & inhibitors, Proto-Oncogene Proteins c-myc genetics, RNA, Messenger biosynthesis, Small Molecule Libraries administration & dosage, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Cell Proliferation drug effects, Neoplasms genetics, Proto-Oncogene Proteins c-myc biosynthesis
- Abstract
We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.
- Published
- 2015
- Full Text
- View/download PDF
8. Mitochondrial proteomics with siRNA knockdown to reveal ACAT1 and MDH2 in the development of doxorubicin-resistant uterine cancer.
- Author
-
Lo YW, Lin ST, Chang SJ, Chan CH, Lyu KW, Chang JF, May EW, Lin DY, Chou HC, and Chan HL
- Subjects
- Acetyl-CoA C-Acetyltransferase genetics, Antibiotics, Antineoplastic pharmacology, Apoptosis drug effects, Cell Line, Tumor, Cell Survival drug effects, Dose-Response Relationship, Drug, Electrophoresis, Gel, Two-Dimensional, Female, Humans, Immunoblotting, Malate Dehydrogenase genetics, Mitochondrial Proteins genetics, Proteome genetics, Proteomics methods, RNA Interference, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Uterine Neoplasms genetics, Uterine Neoplasms metabolism, Uterine Neoplasms pathology, Acetyl-CoA C-Acetyltransferase metabolism, Doxorubicin pharmacology, Drug Resistance, Neoplasm, Malate Dehydrogenase metabolism, Mitochondrial Proteins metabolism, Proteome metabolism
- Abstract
Mitochondria are key organelles in mammary cells in responsible for a number of cellular functions including cell survival and energy metabolism. Moreover, mitochondria are one of the major targets under doxorubicin treatment. In this study, low-abundant mitochondrial proteins were enriched for proteomic analysis with the state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assistant laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the mitochondrial protein profiling changes in response to the development of doxorubicin resistance in human uterine cancer cells. The mitochondrial proteomic results demonstrate more than fifteen hundred protein features were resolved from the equal amount pooled of three purified mitochondrial proteins and 101 differentially expressed spots were identified. In which, 39 out of these 101 identified proteins belong to mitochondrial proteins. Mitochondrial proteins such as acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) have not been reported with the roles on the formation of doxorubicin resistance in our knowledge. Further studies have used RNA interference and cell viability analysis to evidence the essential roles of ACAT1 and MDH2 on their potency in the formation of doxorubicin resistance through increased cell viability and decreased cell apoptosis during doxorubicin treatment. To sum up, our current mitochondrial proteomic approaches allowed us to identify numerous proteins, including ACAT1 and MDH2, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)
- Published
- 2015
- Full Text
- View/download PDF
9. Identification of up- and down-regulated proteins in doxorubicin-resistant uterine cancer cells: reticulocalbin-1 plays a key role in the development of doxorubicin-associated resistance.
- Author
-
May EW, Lin ST, Lin CC, Chang JF, Hung E, Lo YW, Lin LH, Hu RY, Feng CL, Lin DY, Wu SB, Lee WC, Lyu KW, Chou HC, and Chan HL
- Subjects
- Apoptosis drug effects, Calcium-Binding Proteins genetics, Cell Line, Tumor, Down-Regulation drug effects, Electrophoresis, Gel, Two-Dimensional, Female, Humans, Proteome, RNA, Small Interfering administration & dosage, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Up-Regulation drug effects, Antibiotics, Antineoplastic pharmacology, Calcium-Binding Proteins metabolism, Doxorubicin pharmacology, Drug Resistance, Neoplasm physiology, Uterine Neoplasms metabolism
- Abstract
Drug resistance is a frequent cause of failure in cancer chemotherapy treatments. In this study, a pair of uterine sarcoma cancer lines, MES-SA, and doxorubicin-resistant partners, MES-SA/DxR-2μM cells and MES-SA/DxR-8μM cells, as a model system to investigate resistance-dependent proteome alterations and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to perform this research and the results revealed that doxorubicin-resistance altered the expression of 208 proteins in which 129 identified proteins showed dose-dependent manners in response to the levels of resistance. Further studies have used RNA interference, H2A.X phosphorylation assay, cell viability analysis, and analysis of apoptosis against reticulocalbin-1 (RCN1) proteins, to prove its potency on the formation of doxorubicin resistance as well as the attenuation of doxorubicin-associated DNA double strand breakage. To sum up, our results provide useful diagnostic markers and therapeutic candidates such as RCN1 for the treatment of doxorubicin-resistant uterine cancer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
10. Nuclear proteomics with XRCC3 knockdown to reveal the development of doxorubicin-resistant uterine cancer.
- Author
-
Chang JF, Lin ST, Hung E, Lu YL, Soon May EW, Lo YW, Chou HC, and Chan HL
- Subjects
- Cell Line, Tumor, Cell Survival drug effects, DNA-Binding Proteins genetics, Dose-Response Relationship, Drug, Female, Gene Knockdown Techniques, Humans, Protein Array Analysis, RNA, Small Interfering genetics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Transfection, Two-Dimensional Difference Gel Electrophoresis, Uterine Neoplasms genetics, Uterine Neoplasms pathology, Antibiotics, Antineoplastic pharmacology, DNA-Binding Proteins antagonists & inhibitors, Doxorubicin pharmacology, Drug Resistance, Neoplasm genetics, Nuclear Proteins metabolism, Proteomics, Uterine Neoplasms metabolism
- Abstract
The nucleus is a key organelle in mammary cells, which is responsible for several cellular functions including cell proliferation, gene expression, and cell survival. In addition, the nucleus is the primary targets of doxorubicin treatment. In the current study, low-abundance nuclear proteins were enriched for proteomic analysis by using a state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the nuclear protein profiling changes responsible for the development of doxorubicin resistance in human uterine cancer cells. The results of the nuclear proteomic analysis indicated that more than 2100 protein features were resolved from an equal pooled amount of three purified nuclear proteins and 117 differentially expressed spots were identified. Of these 117 identified proteins, 48 belonged to nuclear proteins and a positive correlation was observed between the expression levels of 32 of these nuclear proteins and an increase in drug resistance. According to our review of relevant research, nuclear proteins such as DNA repair protein XRCC3 (XRCC3) have not been reported to play roles in the formation of doxorubicin resistance. Previous studies have used RNA interference and cell viability analysis to evidence the essential roles of XRCC3 on its potency in the formation of doxorubicin resistance. To sum up, our nuclear proteomic approaches enabled us to identify numerous proteins, including XRCC3, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for treating doxorubicin-resistant uterine cancer.
- Published
- 2014
- Full Text
- View/download PDF
11. Discovery of novel insulin-like growth factor-1 receptor inhibitors with unique time-dependent binding kinetics.
- Author
-
Jin M, Petronella BA, Cooke A, Kadalbajoo M, Siu KW, Kleinberg A, May EW, Gokhale PC, Schulz R, Kahler J, Bittner MA, Foreman K, Pachter JA, Wild R, Epstein D, and Mulvihill MJ
- Abstract
This letter describes a series of small molecule inhibitors of IGF-1R with unique time-dependent binding kinetics and slow off-rates. Structure-activity and structure-kinetic relationships were elucidated and guided further optimizations within the series, culminating in compound 2. With an IGF-1R dissociative half-life (t 1/2) of >100 h, compound 2 demonstrated significant and extended PD effects in conjunction with tumor growth inhibition in xenograft models at a remarkably low and intermittent dose, which correlated with the observed in vitro slow off-rate properties.
- Published
- 2013
- Full Text
- View/download PDF
12. Discovery of an Orally Efficacious Imidazo[5,1-f][1,2,4]triazine Dual Inhibitor of IGF-1R and IR.
- Author
-
Jin M, Gokhale PC, Cooke A, Foreman K, Buck E, May EW, Feng L, Bittner MA, Kadalbajoo M, Landfair D, Siu KW, Stolz KM, Werner DS, Laufer RS, Li AH, Dong H, Steinig AG, Kleinberg A, Yao Y, Pachter JA, Wild R, and Mulvihill MJ
- Abstract
This report describes the investigation of a series of 5,7-disubstituted imidazo[5,1-f][1,2,4]triazine inhibitors of insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (IR). Structure-activity relationship exploration and optimization leading to the identification, characterization, and pharmacological activity of compound 9b, a potent, selective, well-tolerated, and orally bioavailable dual inhibitor of IGF-1R and IR with in vivo efficacy in tumor xenograft models, is discussed.
- Published
- 2010
- Full Text
- View/download PDF
13. Type IIA topoisomerase inhibition by a new class of antibacterial agents.
- Author
-
Bax BD, Chan PF, Eggleston DS, Fosberry A, Gentry DR, Gorrec F, Giordano I, Hann MM, Hennessy A, Hibbs M, Huang J, Jones E, Jones J, Brown KK, Lewis CJ, May EW, Saunders MR, Singh O, Spitzfaden CE, Shen C, Shillings A, Theobald AJ, Wohlkonig A, Pearson ND, and Gwynn MN
- Subjects
- Anti-Bacterial Agents metabolism, Apoenzymes chemistry, Apoenzymes metabolism, Arginine metabolism, Aspartic Acid metabolism, Binding Sites, Catalytic Domain, Ciprofloxacin chemistry, Ciprofloxacin metabolism, Crystallography, X-Ray, DNA chemistry, DNA metabolism, DNA Cleavage, DNA Gyrase metabolism, DNA, Superhelical chemistry, DNA, Superhelical metabolism, Drug Design, Drug Resistance, Escherichia coli enzymology, Manganese metabolism, Models, Molecular, Protein Conformation, Quinolines metabolism, Quinolones chemistry, Quinolones metabolism, Structure-Activity Relationship, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, DNA Gyrase chemistry, Quinolines chemistry, Quinolines pharmacology, Staphylococcus aureus enzymology, Topoisomerase II Inhibitors
- Abstract
Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
- Published
- 2010
- Full Text
- View/download PDF
14. An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation.
- Author
-
Rominger CM, Schaber MD, Yang J, Gontarek RR, Weaver KL, Broderick T, Carter L, Copeland RA, and May EW
- Subjects
- Adenosine Triphosphate chemistry, Animals, Butadienes pharmacology, Escherichia coli metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Humans, Hydrolysis, Kinetics, Nitriles pharmacology, Phosphates metabolism, Phosphorylation, Proto-Oncogene Proteins B-raf metabolism, Rabbits, Signal Transduction, Time Factors, Adenosine Triphosphatases metabolism, MAP Kinase Kinase 1 metabolism
- Abstract
We have developed a highly sensitive assay of MEK-mediated ATP hydrolysis by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. Robust ATP hydrolysis is catalyzed by phosphorylated MEK in the absence of the protein substrate ERK. This ERK-uncoupled ATPase activity is dependent on the phosphorylation status of MEK and is abrogated by the selective MEK kinase inhibitor U0126. ADP production is concomitant with Raf-mediated phosphorylation of MEK. Based on this finding, a coupled Raf/MEK assay is developed for measuring the Raf activity. A kinetic treatment derived under steady-state assumptions is presented for the analysis of the reaction progress curve generated by this coupled assay. We have shown that inhibitory potency of selective Raf inhibitors can be determined accurately by this assay.
- Published
- 2007
- Full Text
- View/download PDF
15. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885.
- Author
-
King AJ, Patrick DR, Batorsky RS, Ho ML, Do HT, Zhang SY, Kumar R, Rusnak DW, Takle AK, Wilson DM, Hugger E, Wang L, Karreth F, Lougheed JC, Lee J, Chau D, Stout TJ, May EW, Rominger CM, Schaber MD, Luo L, Lakdawala AS, Adams JL, Contractor RG, Smalley KS, Herlyn M, Morrissey MM, Tuveson DA, and Huang PS
- Subjects
- Alleles, Animals, Blotting, Western, Cell Line, Tumor, Cell Movement drug effects, Cell Proliferation drug effects, Crystallization, Crystallography, X-Ray, Extracellular Signal-Regulated MAP Kinases metabolism, Female, HT29 Cells, Humans, Imidazoles chemistry, Mice, Mice, Nude, Models, Molecular, Molecular Structure, Mutation genetics, Neoplasms enzymology, Neoplasms pathology, Phosphorylation drug effects, Protein Conformation drug effects, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins B-raf chemistry, Proto-Oncogene Proteins B-raf genetics, Xenograft Model Antitumor Assays, Imidazoles therapeutic use, Neoplasms drug therapy, Proto-Oncogene Proteins B-raf antagonists & inhibitors
- Abstract
Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor.
- Published
- 2006
- Full Text
- View/download PDF
16. Unexpected structural diversity in DNA recombination: the restriction endonuclease connection.
- Author
-
Hickman AB, Li Y, Mathew SV, May EW, Craig NL, and Dyda F
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Binding Sites, Catalytic Domain, Crystallography, X-Ray, DNA genetics, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Magnesium metabolism, Models, Molecular, Mutation, Protein Folding, Protein Structure, Secondary, Structure-Activity Relationship, Transposases genetics, Transposases metabolism, Bacterial Proteins chemistry, DNA metabolism, DNA Transposable Elements, DNA-Binding Proteins chemistry, Deoxyribonucleases, Type II Site-Specific chemistry, Escherichia coli Proteins, Recombination, Genetic, Transposases chemistry
- Abstract
Transposition requires a coordinated series of DNA breakage and joining reactions. The Tn7 transposase contains two proteins: TnsA, which carries out DNA breakage at the 5' ends of the transposon, and TnsB, which carries out breakage and joining at the 3' ends of the transposon. TnsB is a member of the retroviral integrase superfamily whose hallmark is a conserved DDE motif. We report here the structure of TnsA at 2.4 A resolution. Surprisingly, the TnsA fold is that of a type II restriction endonuclease. Thus, Tn7 transposition involves a collaboration between polypeptides, one containing a DDE motif and one that does not. This result indicates that the range of biological processes that utilize restriction enzyme-like folds also includes DNA transposition.
- Published
- 2000
- Full Text
- View/download PDF
17. The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products.
- Author
-
Sarnovsky RJ, May EW, and Craig NL
- Subjects
- Amino Acid Sequence, Binding Sites, Cysteine genetics, Cysteine metabolism, DNA Transposable Elements genetics, DNA, Superhelical metabolism, DNA-Binding Proteins chemistry, Electrophoresis, Agar Gel, Integrases metabolism, Manganese pharmacology, Molecular Sequence Data, Mutagenesis, Site-Directed genetics, Mutation genetics, Retroviridae enzymology, Sequence Alignment, Transposases, Bacterial Proteins chemistry, DNA metabolism, DNA Nucleotidyltransferases chemistry
- Abstract
The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.
- Published
- 1996
18. Switching from cut-and-paste to replicative Tn7 transposition.
- Author
-
May EW and Craig NL
- Subjects
- Bacterial Proteins genetics, DNA Repair, DNA-Binding Proteins genetics, Mutagenesis, Site-Directed, Plasmids, Point Mutation, Bacterial Proteins metabolism, DNA Replication, DNA Transposable Elements genetics, DNA-Binding Proteins metabolism, Escherichia coli Proteins, Recombination, Genetic
- Abstract
The bacterial transposon Tn7 usually moves through a cut-and-paste mechanism whereby the transposon is excised from a donor site and joined to a target site to form a simple insertion. The transposon was converted to a replicative element that generated plasmid fusions in vitro and cointegrate products in vivo. This switch was a consequence of the separation of 5'- and 3'-end processing reactions of Tn7 transposition as demonstrated by the consequences of a single amino acid alteration in an element-encoded protein essential for normal cut-and-paste transposition. The mutation specifically blocked cleavage of the 5' strand at each transposon end without disturbing the breakage and joining on the 3' strand, producing a fusion (the Shapiro Intermediate) that resulted in replicative transposition. The ability of Tn7 recombination products to serve as substrates for both the limited gap repair required to complete cut-and-paste transposition and the extensive DNA replication involved in cointegrate formation suggests a remarkable plasticity in Tn7's recruitment of host repair and replication functions.
- Published
- 1996
- Full Text
- View/download PDF
19. A functional analysis of the inverted repeat of the gamma delta transposable element.
- Author
-
May EW and Grindley ND
- Subjects
- Base Sequence, Binding Sites genetics, DNA metabolism, Gene Library, Molecular Sequence Data, Nucleotidyltransferases metabolism, Plasmids, Point Mutation, Protein Binding genetics, Repetitive Sequences, Nucleic Acid, Transposases, DNA genetics, Nucleotidyltransferases genetics
- Abstract
We have constructed a library of point mutants of the 35 base-pair terminal inverted repeat (IR) of the bacterial transposon gamma delta, a member of the Tn3 family of transposable elements. The effect of the mutant ends, both on the immunity conferred on an IR-containing target plasmid and on the transposition of model transposons, was determined. The region important for immunity was shown to be a 30 base-pair stretch of DNA, running from G8 and A9 to G38; mutations in the outermost seven or eight base-pairs did not significantly affect immunity. Positions at which mutations disrupted immunity chiefly coincided with positions previously determined to constitute three segments of the IR with which gamma delta tranposase protein interacts via major groove contacts. We conclude that sequence-specific binding contacts between gamma delta transposase and its cognate IR are limited to a specific subset of positions (those sensitive to mutation in the immunity assay) within this 30 base-pair region. We found that the innermost of the three major groove contact regions was the most susceptible to mutation, while the outermost was the least. Indications of minor groove contacts were also found. Very few point mutations within the 30 base-pair sequence-specific binding region had much effect on transposition when the mutant ends were in the "wild-type" context with the adjacent integration host factor (IHF) binding site. However, deletion of the IHF site, in some cases, revealed a transposition defect, suggesting that for transposition (but not immunity), IHF-transposase cooperation can largely overcome the effects of reduced transposase binding. Although the outer seven base-pairs were not important for immunity, mutations in the outer three or four eliminated or reduced transposition activity, suggesting that these positions are involved in a step in transposition that follows transposase binding.
- Published
- 1995
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.