37 results on '"Leonard PG"'
Search Results
2. Electronic Information: Who Pays the Piper?
- Author
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Conference on Computing Systems and Information Technology (1987 : Brisbane, Qld.) and Leonard, PG
- Published
- 1987
Catalog
3. Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.
- Author
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Hicks KG, Cluntun AA, Schubert HL, Hackett SR, Berg JA, Leonard PG, Ajalla Aleixo MA, Zhou Y, Bott AJ, Salvatore SR, Chang F, Blevins A, Barta P, Tilley S, Leifer A, Guzman A, Arok A, Fogarty S, Winter JM, Ahn HC, Allen KN, Block S, Cardoso IA, Ding J, Dreveny I, Gasper WC, Ho Q, Matsuura A, Palladino MJ, Prajapati S, Sun P, Tittmann K, Tolan DR, Unterlass J, VanDemark AP, Vander Heiden MG, Webb BA, Yun CH, Zhao P, Wang B, Schopfer FJ, Hill CP, Nonato MC, Muller FL, Cox JE, and Rutter J more...
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- Humans, Fatty Acids metabolism, Organ Specificity, Mass Spectrometry methods, Allosteric Regulation, Carbohydrate Metabolism, L-Lactate Dehydrogenase metabolism, Metabolome
- Abstract
Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment. more...
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- 2023
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4. Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site.
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Liu Z, Li Y, Chen H, Lai HT, Wang P, Wu SY, Wold EA, Leonard PG, Joseph S, Hu H, Chiang CM, Brasier AR, Tian B, and Zhou J
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- Animals, Anti-Inflammatory Agents chemical synthesis, Anti-Inflammatory Agents metabolism, Anti-Inflammatory Agents pharmacokinetics, Binding Sites, Cell Cycle Proteins metabolism, Cell Line, Crystallography, X-Ray, Gene Expression drug effects, Humans, Interleukin-6 genetics, Interleukin-6 metabolism, Male, Mice, Inbred C57BL, Oxidoreductases Acting on CH-CH Group Donors genetics, Oxidoreductases Acting on CH-CH Group Donors metabolism, Phenylurea Compounds chemical synthesis, Phenylurea Compounds metabolism, Phenylurea Compounds pharmacokinetics, Protein Binding, Protein Domains, Rats, Transcription Factors metabolism, Mice, Anti-Inflammatory Agents pharmacology, Cell Cycle Proteins antagonists & inhibitors, Nuclear Proteins antagonists & inhibitors, Phenylurea Compounds pharmacology, Transcription Factors antagonists & inhibitors
- Abstract
Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 ( 52 ) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo . The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners. more...
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- 2022
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5. Discovery of 6-[(3 S ,4 S )-4-Amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl]-3-(2,3-dichlorophenyl)-2-methyl-3,4-dihydropyrimidin-4-one (IACS-15414), a Potent and Orally Bioavailable SHP2 Inhibitor.
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Czako B, Sun Y, McAfoos T, Cross JB, Leonard PG, Burke JP, Carroll CL, Feng N, Harris AL, Jiang Y, Kang Z, Kovacs JJ, Mandal P, Meyers BA, Mseeh F, Parker CA, Yu SS, Williams CC, Wu Q, Di Francesco ME, Draetta G, Heffernan T, Marszalek JR, Kohl NE, and Jones P more...
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- Administration, Oral, Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents chemistry, Cell Line, Tumor, Cell Proliferation drug effects, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, Enzyme Inhibitors administration & dosage, Enzyme Inhibitors chemistry, Humans, Mice, Molecular Structure, Neoplasms, Experimental drug therapy, Neoplasms, Experimental metabolism, Neoplasms, Experimental pathology, Protein Tyrosine Phosphatase, Non-Receptor Type 11 metabolism, Structure-Activity Relationship, Antineoplastic Agents pharmacology, Drug Discovery, Enzyme Inhibitors pharmacology, Protein Tyrosine Phosphatase, Non-Receptor Type 11 antagonists & inhibitors
- Abstract
Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-à-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 μM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRAS
mut xenograft models in vivo. more...- Published
- 2021
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6. Discovery of IACS-9779 and IACS-70465 as Potent Inhibitors Targeting Indoleamine 2,3-Dioxygenase 1 (IDO1) Apoenzyme.
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Hamilton MM, Mseeh F, McAfoos TJ, Leonard PG, Reyna NJ, Harris AL, Xu A, Han M, Soth MJ, Czako B, Theroff JP, Mandal PK, Burke JP, Virgin-Downey B, Petrocchi A, Pfaffinger D, Rogers NE, Parker CA, Yu SS, Jiang Y, Krapp S, Lammens A, Trevitt G, Tremblay MR, Mikule K, Wilcoxen K, Cross JB, Jones P, Marszalek JR, and Lewis RT more...
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- Dose-Response Relationship, Drug, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors chemistry, Humans, Indoleamine-Pyrrole 2,3,-Dioxygenase metabolism, Molecular Structure, Structure-Activity Relationship, Drug Discovery, Enzyme Inhibitors pharmacology, Indoleamine-Pyrrole 2,3,-Dioxygenase antagonists & inhibitors
- Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 ( 62 ) and IACS-70465 ( 71 ). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development. more...
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- 2021
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7. GRB2 enforces homology-directed repair initiation by MRE11.
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Ye Z, Xu S, Shi Y, Bacolla A, Syed A, Moiani D, Tsai CL, Shen Q, Peng G, Leonard PG, Jones DE, Wang B, Tainer JA, and Ahmed Z
- Abstract
DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).) more...
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- 2021
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8. Author Correction: An enolase inhibitor for the targeted treatment of ENO1-deleted cancers.
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Lin YH, Satani N, Hammoudi N, Yan VC, Barekatain Y, Khadka S, Ackroyd JJ, Georgiou DK, Pham CD, Arthur K, Maxwell D, Peng Z, Leonard PG, Czako B, Pisaneschi F, Mandal P, Sun Y, Zielinski R, Pando SC, Wang X, Tran T, Xu Q, Wu Q, Jiang Y, Kang Z, Asara JM, Priebe W, Bornmann W, Marszalek JR, DePinho RA, and Muller FL more...
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- 2021
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9. An enolase inhibitor for the targeted treatment of ENO1-deleted cancers.
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Lin YH, Satani N, Hammoudi N, Yan VC, Barekatain Y, Khadka S, Ackroyd JJ, Georgiou DK, Pham CD, Arthur K, Maxwell D, Peng Z, Leonard PG, Czako B, Pisaneschi F, Mandal P, Sun Y, Zielinski R, Pando SC, Wang X, Tran T, Xu Q, Wu Q, Jiang Y, Kang Z, Asara JM, Priebe W, Bornmann W, Marszalek JR, DePinho RA, and Muller FL more...
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- Animals, Cell Line, Tumor, Female, Glioma drug therapy, Glycolysis drug effects, Humans, Macaca fascicularis, Male, Mice, Mice, SCID, Phosphopyruvate Hydratase genetics, Precision Medicine, Sequence Deletion, Structure-Activity Relationship, Xenograft Model Antitumor Assays, Antineoplastic Agents therapeutic use, Biomarkers, Tumor genetics, DNA-Binding Proteins genetics, Enzyme Inhibitors therapeutic use, Neoplasms drug therapy, Neoplasms genetics, Phosphopyruvate Hydratase antagonists & inhibitors, Tumor Suppressor Proteins genetics
- Abstract
Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings. more...
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- 2020
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10. Discovery of IPN60090, a Clinical Stage Selective Glutaminase-1 (GLS-1) Inhibitor with Excellent Pharmacokinetic and Physicochemical Properties.
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Soth MJ, Le K, Di Francesco ME, Hamilton MM, Liu G, Burke JP, Carroll CL, Kovacs JJ, Bardenhagen JP, Bristow CA, Cardozo M, Czako B, de Stanchina E, Feng N, Garvey JR, Gay JP, Do MKG, Greer J, Han M, Harris A, Herrera Z, Huang S, Giuliani V, Jiang Y, Johnson SB, Johnson TA, Kang Z, Leonard PG, Liu Z, McAfoos T, Miller M, Morlacchi P, Mullinax RA, Palmer WS, Pang J, Rogers N, Rudin CM, Shepard HE, Spencer ND, Theroff J, Wu Q, Xu A, Yau JA, Draetta G, Toniatti C, Heffernan TP, and Jones P more...
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- Administration, Oral, Animals, Cell Line, Tumor, Dogs, Drug Evaluation, Preclinical, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacokinetics, Glutaminase genetics, Glutaminase metabolism, Half-Life, Hepatocytes cytology, Hepatocytes drug effects, Hepatocytes metabolism, Humans, Inhibitory Concentration 50, Male, Mice, Microsomes metabolism, Protein Binding, Rats, Rats, Sprague-Dawley, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Structure-Activity Relationship, Triazoles chemistry, Triazoles metabolism, Enzyme Inhibitors chemistry, Glutaminase antagonists & inhibitors, Triazoles pharmacokinetics
- Abstract
Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans. more...
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- 2020
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11. Discovery of IACS-9439, a Potent, Exquisitely Selective, and Orally Bioavailable Inhibitor of CSF1R.
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Czako B, Marszalek JR, Burke JP, Mandal P, Leonard PG, Cross JB, Mseeh F, Jiang Y, Chang EQ, Suzuki E, Kovacs JJ, Feng N, Gera S, Harris AL, Liu Z, Mullinax RA, Pang J, Parker CA, Spencer ND, Yu SS, Wu Q, Tremblay MR, Mikule K, Wilcoxen K, Heffernan TP, Draetta GF, and Jones P more...
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- Antineoplastic Agents chemical synthesis, Antineoplastic Agents pharmacokinetics, Benzothiazoles chemical synthesis, Benzothiazoles pharmacokinetics, Drug Stability, Humans, Microsomes, Liver metabolism, Molecular Structure, Pyrimidines chemical synthesis, Pyrimidines pharmacokinetics, Structure-Activity Relationship, THP-1 Cells, Tumor-Associated Macrophages drug effects, Antineoplastic Agents therapeutic use, Benzothiazoles therapeutic use, Neoplasms drug therapy, Pyrimidines therapeutic use, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor antagonists & inhibitors
- Abstract
Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 ( 1 ). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models. more...
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- 2020
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12. Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological Evaluation.
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Liu Z, Chen H, Wang P, Li Y, Wold EA, Leonard PG, Joseph S, Brasier AR, Tian B, and Zhou J
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- Administration, Oral, Animals, Biological Availability, Cell Cycle Proteins metabolism, Cell Line, Transformed, Chromones pharmacology, Crystallization methods, Crystallization trends, Drug Discovery trends, Drug Evaluation, Preclinical methods, Humans, Male, Mice, Mice, Inbred C57BL, Protein Structure, Secondary, Protein Structure, Tertiary, Structure-Activity Relationship, Transcription Factors metabolism, Cell Cycle Proteins antagonists & inhibitors, Chromones administration & dosage, Chromones chemistry, Drug Discovery methods, Transcription Factors antagonists & inhibitors
- Abstract
Bromodomain-containing protein 4 (BRD4) represents a promising drug target for anti-inflammatory therapeutics. Herein, we report the design, synthesis, and pharmacological evaluation of novel chromone derivatives via scaffold hopping to discover a new class of orally bioavailable BRD4-selective inhibitors. Two potent BRD4 bromodomain 1 (BD1)-selective inhibitors 44 (ZL0513) and 45 (ZL0516) have been discovered with high binding affinity (IC
50 values of 67-84 nM) and good selectivity over other BRD family proteins and distant BD-containing proteins. Both compounds significantly inhibited the expression of Toll-like receptor-induced inflammatory genes in vitro and airway inflammation in murine models. The cocrystal structure of 45 in complex with human BRD4 BD1 at a high resolution of 2.0 Å has been solved, offering a solid structural basis for its binding validation and further structure-based optimization. These BRD4 BD1 inhibitors demonstrated impressive in vivo efficacy and overall promising pharmacokinetic properties, indicating their therapeutic potential for the treatment of inflammatory diseases. more...- Published
- 2020
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13. The 3 S Enantiomer Drives Enolase Inhibitory Activity in SF2312 and Its Analogues.
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Pisaneschi F, Lin YH, Leonard PG, Satani N, Yan VC, Hammoudi N, Raghavan S, Link TM, K Georgiou D, Czako B, and Muller FL
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- Binding Sites, Enzyme Activation drug effects, Enzyme Inhibitors chemistry, Models, Molecular, Molecular Conformation, Molecular Structure, Organophosphonates chemistry, Protein Binding, Pyrrolidinones chemistry, Spectrum Analysis, Stereoisomerism, Structure-Activity Relationship, Enzyme Inhibitors pharmacology, Organophosphonates pharmacology, Phosphopyruvate Hydratase antagonists & inhibitors, Phosphopyruvate Hydratase chemistry, Pyrrolidinones pharmacology
- Abstract
We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3 S ,5 S )-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3 S )-MethylSF2312 was up to 2000-fold more potent than (3 R )-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3 S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3 S, 5 S) -enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3 S, 5 S) -enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3 S ,5 S )-SF2312 is the single active enantiomer of inhibitor SF2312. more...
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- 2019
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14. Neomorphic PDGFRA extracellular domain driver mutations are resistant to PDGFRA targeted therapies.
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Ip CKM, Ng PKS, Jeong KJ, Shao SH, Ju Z, Leonard PG, Hua X, Vellano CP, Woessner R, Sahni N, Scott KL, and Mills GB
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- Animals, Cell Line, Cell Proliferation, Endoplasmic Reticulum metabolism, Glycosylation, Golgi Apparatus metabolism, Humans, Mice, Phenotype, Protein Domains, Protein Kinase Inhibitors pharmacology, Signal Transduction, Drug Resistance, Neoplasm genetics, Extracellular Space chemistry, Molecular Targeted Therapy, Mutation genetics, Receptor, Platelet-Derived Growth Factor alpha chemistry, Receptor, Platelet-Derived Growth Factor alpha genetics
- Abstract
Activation of platelet-derived growth factor receptor alpha (PDGFRA) by genomic aberrations contributes to tumor progression in several tumor types. In this study, we characterize 16 novel PDGFRA mutations identified from different tumor types and identify three previously uncharacterized activating mutations that promote cell survival and proliferation. PDGFRA Y288C, an extracellular domain mutation, is primarily high mannose glycosylated consistent with trapping in the endoplasmic reticulum (ER). Strikingly, PDGFRA Y288C is constitutively dimerized and phosphorylated in the absence of ligand suggesting that trapping in the ER or aberrant glycosylation is sufficient for receptor activation. Importantly, PDGFRA Y288C induces constitutive phosphorylation of Akt, ERK1/2, and STAT3. PDGFRA Y288C is resistant to PDGFR inhibitors but sensitive to PI3K/mTOR and MEK inhibitors consistent with pathway activation results. Our findings further highlight the importance of characterizing functional consequences of individual mutations for precision medicine. more...
- Published
- 2018
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15. The N-Terminal Domain of ALS-Linked TDP-43 Assembles without Misfolding.
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Tsoi PS, Choi KJ, Leonard PG, Sizovs A, Moosa MM, MacKenzie KR, Ferreon JC, and Ferreon ACM
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- Humans, Hydrogen-Ion Concentration, Models, Molecular, Protein Aggregation, Pathological pathology, Protein Domains, Protein Multimerization, Thermodynamics, Amyotrophic Lateral Sclerosis pathology, DNA-Binding Proteins chemistry, Protein Folding
- Abstract
Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) misfolding is implicated in several neurodegenerative diseases characterized by aggregated protein inclusions. Misfolding is believed to be mediated by both the N- and C-terminus of TDP-43; however, the mechanistic basis of the contribution of individual domains in the process remained elusive. Here, using single-molecule fluorescence and ensemble biophysical techniques, and a wide range of pH and temperature conditions, we show that TDP-43
NTD is thermodynamically stable, well-folded and undergoes reversible oligomerization. We propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...- Published
- 2017
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16. SF2312 is a natural phosphonate inhibitor of enolase.
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Leonard PG, Satani N, Maxwell D, Lin YH, Hammoudi N, Peng Z, Pisaneschi F, Link TM, Lee GR 4th, Sun D, Prasad BAB, Di Francesco ME, Czako B, Asara JM, Wang YA, Bornmann W, DePinho RA, and Muller FL
- Subjects
- Dose-Response Relationship, Drug, Enzyme Inhibitors chemistry, Humans, Models, Molecular, Molecular Structure, Organophosphonates chemistry, Phosphopyruvate Hydratase metabolism, Pyrrolidinones chemistry, Structure-Activity Relationship, Enzyme Inhibitors pharmacology, Organophosphonates pharmacology, Phosphopyruvate Hydratase antagonists & inhibitors, Pyrrolidinones pharmacology
- Abstract
Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase. more...
- Published
- 2016
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17. An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.
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Davlieva M, Tovar-Yanez A, DeBruler K, Leonard PG, Zianni MR, Arias CA, and Shamoo Y
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- Adaptation, Biological, Bacterial Proteins chemistry, Bacterial Proteins genetics, Crystallography, X-Ray, DNA, Bacterial metabolism, Enterococcus faecium genetics, Gene Expression Regulation, Bacterial, Magnetic Resonance Spectroscopy, Models, Molecular, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins metabolism, Operon, Phosphorylation, Promoter Regions, Genetic, Protein Binding, Protein Conformation, Protein Multimerization, Protein Processing, Post-Translational, Transcription Factors chemistry, Transcription Factors genetics, Transcription, Genetic, Anti-Bacterial Agents pharmacology, Bacterial Proteins metabolism, Daptomycin pharmacology, Drug Resistance, Bacterial, Enterococcus faecium drug effects, Mutation, Transcription Factors metabolism
- Abstract
The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaR
W73C ). We have determined the structure of the activated E. faecium LiaR protein at 3.2Å resolution and, in combination with solution studies, show that the activation of LiaR induces the formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to DAP by the upregulation of the LiaFSR regulon. A comparison of the E. faecalis LiaR, E. faecium LiaR, and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with DAP resistance suggests that physicochemical properties such as oligomerization state and DNA specificity, although tuned to the biology of each organism, share some features that could be targeted for new antimicrobials., (Copyright © 2016 Elsevier Ltd. All rights reserved.) more...- Published
- 2016
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18. Corrigendum: Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors.
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Du Y, Yamaguchi H, Wei Y, Hsu JL, Wang HL, Hsu YH, Lin WC, Yu WH, Leonard PG, Lee GR 4th, Chen MK, Nakai K, Hsu MC, Chen CT, Sun Y, Wu Y, Chang WC, Huang WC, Liu CL, Chang YC, Chen CH, Park M, Jones P, Hortobagyi GN, and Hung MC more...
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- 2016
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19. Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors.
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Du Y, Yamaguchi H, Wei Y, Hsu JL, Wang HL, Hsu YH, Lin WC, Yu WH, Leonard PG, Lee GR 4th, Chen MK, Nakai K, Hsu MC, Chen CT, Sun Y, Wu Y, Chang WC, Huang WC, Liu CL, Chang YC, Chen CH, Park M, Jones P, Hortobagyi GN, and Hung MC more...
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- Anilides pharmacology, Animals, Benzimidazoles pharmacology, Cell Line, Tumor, Cell Survival drug effects, Crizotinib, Humans, In Vitro Techniques, Indoles pharmacology, MCF-7 Cells, Mice, Neoplasm Transplantation, Phosphorylation drug effects, Phthalazines pharmacology, Piperazines pharmacology, Poly (ADP-Ribose) Polymerase-1, Poly(ADP-ribose) Polymerases drug effects, Pyrazoles pharmacology, Pyridines pharmacology, Quinolines pharmacology, Xenograft Model Antitumor Assays, Antineoplastic Combined Chemotherapy Protocols pharmacology, Apoptosis drug effects, Breast Neoplasms, Cell Proliferation drug effects, Lung Neoplasms, Poly(ADP-ribose) Polymerase Inhibitors pharmacology, Poly(ADP-ribose) Polymerases metabolism, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-met antagonists & inhibitors
- Abstract
Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials. One PARP inhibitor, olaparib (Lynparza, AstraZeneca), was recently approved by the FDA to treat ovarian cancer with mutations in BRCA genes. BRCA1 and BRCA2 have essential roles in repairing DNA double-strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition. Here we show that the receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907 (PARP1 pTyr907 or pY907). PARP1 pY907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. The combination of c-Met and PARP1 inhibitors synergized to suppress the growth of breast cancer cells in vitro and xenograft tumor models, and we observed similar synergistic effects in a lung cancer xenograft tumor model. These results suggest that the abundance of PARP1 pY907 may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients whose tumors show high c-Met expression and who do not respond to PARP inhibition alone. more...
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- 2016
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20. Potent and selective inhibition of SH3 domains with dirhodium metalloinhibitors.
- Author
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Vohidov F, Knudsen SE, Leonard PG, Ohata J, Wheadon MJ, Popp BV, Ladbury JE, and Ball ZT
- Abstract
Src-family kinases (SFKs) play important roles in human biology and are key drug targets as well. However, achieving selective inhibition of individual Src-family kinases is challenging due to the high similarity within the protein family. We describe rhodium(ii) conjugates that deliver both potent and selective inhibition of Src-family SH3 domains. Rhodium(ii) conjugates offer dramatic affinity enhancements due to interactions with specific and unique Lewis-basic histidine residues near the SH3 binding interface, allowing predictable, structure-guided inhibition of SH3 targets that are recalcitrant to traditional inhibitors. In one example, a simple metallopeptide binds the Lyn SH3 domain with 6 nM affinity and exhibits functional activation of Lyn kinase under biologically relevant concentrations (EC
50 ∼ 200 nM). more...- Published
- 2015
- Full Text
- View/download PDF
21. A variable DNA recognition site organization establishes the LiaR-mediated cell envelope stress response of enterococci to daptomycin.
- Author
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Davlieva M, Shi Y, Leonard PG, Johnson TA, Zianni MR, Arias CA, Ladbury JE, and Shamoo Y
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, Consensus Sequence, DNA, Bacterial metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Drug Resistance, Bacterial, Enterococcus faecalis drug effects, Enterococcus faecalis genetics, Models, Molecular, Mutation, Operon, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Anti-Bacterial Agents pharmacology, Bacterial Proteins chemistry, DNA, Bacterial chemistry, DNA-Binding Proteins chemistry, Daptomycin pharmacology
- Abstract
LiaR is a 'master regulator' of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR(D191N) increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.) more...
- Published
- 2015
- Full Text
- View/download PDF
22. Observed bromodomain flexibility reveals histone peptide- and small molecule ligand-compatible forms of ATAD2.
- Author
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Poncet-Montange G, Zhan Y, Bardenhagen JP, Petrocchi A, Leo E, Shi X, Lee GR 4th, Leonard PG, Geck Do MK, Cardozo MG, Andersen JN, Palmer WS, Jones P, and Ladbury JE
- Subjects
- ATPases Associated with Diverse Cellular Activities, Adenosine Triphosphatases antagonists & inhibitors, Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, Antineoplastic Agents chemical synthesis, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Binding Sites, Biotinylation, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, Histones antagonists & inhibitors, Histones metabolism, Humans, Isoxazoles chemical synthesis, Isoxazoles pharmacology, Kinetics, Ligands, Mutant Proteins antagonists & inhibitors, Mutant Proteins chemistry, Mutant Proteins metabolism, Peptide Fragments antagonists & inhibitors, Peptide Fragments metabolism, Pliability, Protein Conformation, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sulfonamides chemical synthesis, Sulfonamides chemistry, Sulfonamides pharmacology, meta-Aminobenzoates chemical synthesis, meta-Aminobenzoates chemistry, meta-Aminobenzoates pharmacology, Adenosine Triphosphatases chemistry, DNA-Binding Proteins chemistry, Drug Design, Enzyme Inhibitors chemistry, Histones chemistry, Isoxazoles chemistry, Peptide Fragments chemistry, Protein Processing, Post-Translational
- Abstract
Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies. more...
- Published
- 2015
- Full Text
- View/download PDF
23. The human orphan nuclear receptor tailless (TLX, NR2E1) is druggable.
- Author
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Benod C, Villagomez R, Filgueira CS, Hwang PK, Leonard PG, Poncet-Montange G, Rajagopalan S, Fletterick RJ, Gustafsson JÅ, and Webb P
- Subjects
- Amino Acid Sequence, Binding Sites, COUP Transcription Factor II antagonists & inhibitors, COUP Transcription Factor II physiology, Estrogen Receptor beta antagonists & inhibitors, Estrogen Receptor beta physiology, Genes, Reporter, HeLa Cells, Humans, Inhibitory Concentration 50, Ligands, Luciferases, Renilla biosynthesis, Luciferases, Renilla genetics, Models, Molecular, Molecular Sequence Data, Orphan Nuclear Receptors, Protein Binding, Receptors, Cytoplasmic and Nuclear antagonists & inhibitors, Receptors, Cytoplasmic and Nuclear chemistry, Retinoid X Receptor alpha antagonists & inhibitors, Retinoid X Receptor alpha physiology, Transcription, Genetic drug effects, Dydrogesterone pharmacology, Piperazines pharmacology, Pyrazoles pharmacology, Receptors, Cytoplasmic and Nuclear physiology, Transcriptional Activation drug effects
- Abstract
Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open. more...
- Published
- 2014
- Full Text
- View/download PDF
24. (19)F NMR reveals multiple conformations at the dimer interface of the nonstructural protein 1 effector domain from influenza A virus.
- Author
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Aramini JM, Hamilton K, Ma LC, Swapna GVT, Leonard PG, Ladbury JE, Krug RM, and Montelione GT
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, Fluorine-19 Magnetic Resonance Imaging, Gene Expression, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Multimerization, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Tryptophan chemistry, Viral Nonstructural Proteins genetics, Influenza A Virus, H3N2 Subtype chemistry, Molecular Dynamics Simulation, RNA, Double-Stranded chemistry, Tryptophan analogs & derivatives, Viral Nonstructural Proteins chemistry
- Abstract
Nonstructural protein 1 of influenza A virus (NS1A) is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply (19)F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan, and we demonstrate that the (19)F signal of Trp187 is a sensitive, direct monitor of the ED helix:helix dimer interface. (19)F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein:protein interface, whose rate is more than three orders of magnitude faster than the kinetics of ED dimerization. (19)F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1A at concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.) more...
- Published
- 2014
- Full Text
- View/download PDF
25. FAK dimerization controls its kinase-dependent functions at focal adhesions.
- Author
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Brami-Cherrier K, Gervasi N, Arsenieva D, Walkiewicz K, Boutterin MC, Ortega A, Leonard PG, Seantier B, Gasmi L, Bouceba T, Kadaré G, Girault JA, and Arold ST
- Subjects
- Amino Acid Motifs, Animals, Crystallography, X-Ray, Dimerization, Enzyme Activation, Focal Adhesion Kinase 1 physiology, Focal Adhesions, HEK293 Cells, Humans, Models, Molecular, Phosphorylation, Phosphotyrosine physiology, Protein Conformation, Protein Processing, Post-Translational, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins chemistry, Scattering, Radiation, Focal Adhesion Kinase 1 chemistry
- Abstract
Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions--autophosphorylation of tyrosine-397--requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. more...
- Published
- 2014
- Full Text
- View/download PDF
26. Competition between Grb2 and Plcγ1 for FGFR2 regulates basal phospholipase activity and invasion.
- Author
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Timsah Z, Ahmed Z, Lin CC, Melo FA, Stagg LJ, Leonard PG, Jeyabal P, Berrout J, O'Neil RG, Bogdanov M, and Ladbury JE
- Subjects
- Binding Sites, Binding, Competitive, Cell Line, Tumor, GRB2 Adaptor Protein metabolism, HEK293 Cells, Humans, Models, Genetic, Neoplasm Invasiveness genetics, Phospholipase C gamma metabolism, Protein Structure, Tertiary, GRB2 Adaptor Protein physiology, Phospholipase C gamma physiology, Phospholipases physiology, Receptor, Fibroblast Growth Factor, Type 2 metabolism
- Abstract
FGFR2-expressing human cancer cells with low concentrations of the adaptor protein Grb2 show high prevalence for metastatic outcome. In nonstimulated cells, the SH3 domain (and not the SH2 domains) of Plcγ1 directly competes for a binding site at the very C terminus of FGFR2 with the C-terminal SH3 domain of Grb2. Reduction of Grb2 concentration permits Plcγ1 access to the receptor. Recruitment of Plcγ1 in this way is sufficient to upregulate phospholipase activity. This results in elevated phosphatidylinositol 4,5-bisphosphate turnover and intracellular calcium levels, thus leading to increased cell motility and promotion of cell-invasive behavior in the absence of extracellular receptor stimulation. Therefore, metastatic outcome can be dictated by the constitutive competition between Grb2 and Plcγ1 for the phosphorylation-independent binding site on FGFR2. more...
- Published
- 2014
- Full Text
- View/download PDF
27. Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation.
- Author
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Leonard PG, Golemi-Kotra D, and Stock AM
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Crystallography, X-Ray, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Drug Resistance, Bacterial physiology, Phosphorylation physiology, Protein Structure, Quaternary, Protein Structure, Tertiary, Staphylococcus aureus genetics, Staphylococcus aureus metabolism, Bacterial Proteins chemistry, DNA-Binding Proteins chemistry, Protein Multimerization, Staphylococcus aureus chemistry
- Abstract
Staphylococcus aureus VraR, a vancomycin-resistance-associated response regulator, activates a cell-wall-stress stimulon in response to antibiotics that inhibit cell wall formation. X-ray crystal structures of VraR in both unphosphorylated and beryllofluoride-activated states have been determined, revealing a mechanism of phosphorylation-induced dimerization that features a deep hydrophobic pocket at the center of the receiver domain interface. Unphosphorylated VraR exists in a closed conformation that inhibits dimer formation. Phosphorylation at the active site promotes conformational changes that are propagated throughout the receiver domain, promoting the opening of a hydrophobic pocket that is essential for homodimer formation and enhanced DNA-binding activity. This prominent feature in the VraR dimer can potentially be exploited for the development of novel therapeutics to counteract antibiotic resistance in this important pathogen. more...
- Published
- 2013
- Full Text
- View/download PDF
28. Identification of a hydrophobic cleft in the LytTR domain of AgrA as a locus for small molecule interactions that inhibit DNA binding.
- Author
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Leonard PG, Bezar IF, Sidote DJ, and Stock AM
- Subjects
- Bacterial Proteins antagonists & inhibitors, Crystallization, Crystallography, X-Ray, Hydrophobic and Hydrophilic Interactions, Molecular Docking Simulation, Nuclear Magnetic Resonance, Biomolecular, Peptide Library, Peptides, Cyclic antagonists & inhibitors, Protein Structure, Tertiary drug effects, Quorum Sensing drug effects, Staphylococcus aureus genetics, Bacterial Proteins chemistry, DNA-Binding Proteins chemistry, Peptides, Cyclic chemistry
- Abstract
The AgrA transcription factor regulates the quorum-sensing response in Staphylococcus aureus, controlling the production of hemolysins and other virulence factors. AgrA binds to DNA via its C-terminal LytTR domain, a domain not found in humans but common in many pathogenic bacteria, making it a potential target for antimicrobial development. We have determined the crystal structure of the apo AgrA LytTR domain and screened a library of 500 fragment compounds to find inhibitors of AgrA DNA binding activity. Using nuclear magnetic resonance, the binding site for five compounds has been mapped to a common locus at the C-terminal end of the LytTR domain, a site known to be important for DNA binding activity. Three of these compounds inhibit AgrA DNA binding. These results provide the first evidence that LytTR domains can be targeted by small organic compounds. more...
- Published
- 2012
- Full Text
- View/download PDF
29. Human cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) is dimeric in its disulfide-reduced state, with natively disordered N-terminal region.
- Author
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Ertekin A, Aramini JM, Rossi P, Leonard PG, Janjua H, Xiao R, Maglaqui M, Lee HW, Prestegard JH, and Montelione GT
- Subjects
- Animals, Cell Line, Tumor, Cricetinae, Cyclin-Dependent Kinase 2 chemistry, Cyclin-Dependent Kinase 2 genetics, Cyclin-Dependent Kinase 2 metabolism, Disulfides chemistry, Disulfides metabolism, G1 Phase physiology, Humans, Nuclear Magnetic Resonance, Biomolecular, Oxidation-Reduction, Protein Structure, Quaternary, Protein Structure, Tertiary, S Phase physiology, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, Protein Multimerization, Tumor Suppressor Proteins chemistry
- Abstract
CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), corresponding to the gene doc-1 (deleted in oral cancer 1), is a tumor suppressor protein. The doc-1 gene is absent or down-regulated in hamster oral cancer cells and in many other cancer cell types. The ubiquitously expressed CDK2AP1 protein is the only known specific inhibitor of CDK2, making it an important component of cell cycle regulation during G(1)-to-S phase transition. Here, we report the solution structure of CDK2AP1 by combined methods of solution state NMR and amide hydrogen/deuterium exchange measurements with mass spectrometry. The homodimeric structure of CDK2AP1 includes an intrinsically disordered 60-residue N-terminal region and a four-helix bundle dimeric structure with reduced Cys-105 in the C-terminal region. The Cys-105 residues are, however, poised for disulfide bond formation. CDK2AP1 is phosphorylated at a conserved Ser-46 site in the N-terminal "intrinsically disordered" region by IκB kinase ε. more...
- Published
- 2012
- Full Text
- View/download PDF
30. Structural basis for the sequence-specific recognition of human ISG15 by the NS1 protein of influenza B virus.
- Author
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Guan R, Ma LC, Leonard PG, Amer BR, Sridharan H, Zhao C, Krug RM, and Montelione GT
- Subjects
- Binding Sites, Crystallography, X-Ray, Humans, Interferons pharmacology, Protein Binding, Protein Interaction Domains and Motifs, Cytokines metabolism, Influenza B virus chemistry, Ubiquitins metabolism, Viral Nonstructural Proteins metabolism
- Abstract
Interferon-induced ISG15 conjugation plays an important antiviral role against several viruses, including influenza viruses. The NS1 protein of influenza B virus (NS1B) specifically binds only human and nonhuman primate ISG15s and inhibits their conjugation. To elucidate the structural basis for the sequence-specific recognition of human ISG15, we determined the crystal structure of the complex formed between human ISG15 and the N-terminal region of NS1B (NS1B-NTR). The NS1B-NTR homodimer interacts with two ISG15 molecules in the crystal and also in solution. The two ISG15-binding sites on the NS1B-NTR dimer are composed of residues from both chains, namely residues in the RNA-binding domain (RBD) from one chain, and residues in the linker between the RBD and the effector domain from the other chain. The primary contact region of NS1B-NTR on ISG15 is composed of residues at the junction of the N-terminal ubiquitin-like (Ubl) domain and the short linker region between the two Ubl domains, explaining why the sequence of the short linker in human and nonhuman primate ISG15s is essential for the species-specific binding of these ISG15s. In addition, the crystal structure identifies NS1B-NTR binding sites in the N-terminal Ubl domain of ISG15, and shows that there are essentially no contacts with the C-terminal Ubl domain of ISG15. Consequently, NS1B-NTR binding to ISG15 would not occlude access of the C-terminal Ubl domain of ISG15 to its conjugating enzymes. Nonetheless, transfection assays show that NS1B-NTR binding of ISG15 is responsible for the inhibition of interferon-induced ISG15 conjugation in cells. more...
- Published
- 2011
- Full Text
- View/download PDF
31. H-NS forms a superhelical protein scaffold for DNA condensation.
- Author
-
Arold ST, Leonard PG, Parkinson GN, and Ladbury JE
- Subjects
- Amino Acid Sequence, Biopolymers chemistry, Models, Molecular, Molecular Sequence Data, Sequence Homology, Amino Acid, Temperature, Bacterial Proteins chemistry, DNA chemistry, DNA-Binding Proteins chemistry
- Abstract
The histone-like nucleoid structuring (H-NS) protein plays a fundamental role in DNA condensation and is a key regulator of enterobacterial gene expression in response to changes in osmolarity, pH, and temperature. The protein is capable of high-order self-association via interactions of its oligomerization domain. Using crystallography, we have solved the structure of this complete domain in an oligomerized state. The observed superhelical structure establishes a mechanism for the self-association of H-NS via both an N-terminal antiparallel coiled-coil and a second, hitherto unidentified, helix-turn-helix dimerization interface at the C-terminal end of the oligomerization domain. The helical scaffold suggests the formation of a H-NS:plectonemic DNA nucleoprotein complex that is capable of explaining published biophysical and functional data, and establishes a unifying structural basis for coordinating the DNA packaging and transcription repression functions of H-NS. more...
- Published
- 2010
- Full Text
- View/download PDF
32. The absence of inorganic salt is required for the crystallization of the complete oligomerization domain of Salmonella typhimurium histone-like nucleoid-structuring protein.
- Author
-
Leonard PG, Parkinson GN, Gor J, Perkins SJ, and Ladbury JE
- Subjects
- Bacterial Proteins metabolism, Crystallization, Crystallography, X-Ray, Histones metabolism, Osmolar Concentration, Salmonella typhimurium metabolism, Bacterial Proteins chemistry, Histones chemistry, Protein Multimerization, Salmonella typhimurium chemistry
- Abstract
The histone-like nucleoid-structuring protein (H-NS) plays an important role in both DNA packaging and global gene regulation in enterobacteria. Self-association of the N-terminal domain results in polydisperse oligomers that are critical to the function of the protein. This heterogeneity in oligomer size has so far prevented structure determination of the complete oligomerization domain by NMR or X-ray crystallography. In the absence of inorganic salt, the H-NS oligomerization domain is predominantly restricted to an equilibrium between a homodimer and homotetramer, allowing a protein solution to be prepared that is sufficiently homogeneous for successful crystallization. Crystallization was achieved by tailoring the conditions screened to those identified as minimizing the potential disruption of protein-solution homogeneity. This finding provides a significant step towards resolving the structure of this important prokaryotic protein. more...
- Published
- 2010
- Full Text
- View/download PDF
33. Investigation of the self-association and hetero-association interactions of H-NS and StpA from Enterobacteria.
- Author
-
Leonard PG, Ono S, Gor J, Perkins SJ, and Ladbury JE
- Subjects
- Bacterial Proteins genetics, Chromatography, Gel, DNA-Binding Proteins genetics, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Molecular Chaperones genetics, Mutagenesis, Site-Directed, Protein Multimerization, Protein Stability, Salmonella typhimurium genetics, Salmonella typhimurium metabolism, Bacterial Proteins metabolism, DNA-Binding Proteins metabolism, Escherichia coli Proteins metabolism, Molecular Chaperones metabolism
- Abstract
The nucleoid-associated protein H-NS and its paralogue StpA are global regulators of gene expression and form an integral part of the protein scaffold responsible for DNA condensation in Escherichia coli and Salmonella typhimurium. Although protein oligomerization is a requirement for this function, it is not entirely understood how this is accomplished. We address this by reporting on the self-association of H-NS and its hetero-association with StpA. We identify residues 1-77 of H-NS as being necessary and sufficient for high-order association. A multi-technique-based approach was used to measure the effects of salt concentration on the size distribution of H-NS and the thermal stability of H-NS and StpA dimers. The thermal stability of the StpA homodimer is significantly greater than that of H-NS(1-74). Investigation of the hetero-association of H-NS and StpA proteins suggested that the association of H-NS with StpA is more stable than the self-association of either H-NS or StpA with themselves. This provides a clear understanding of the method of oligomerization of these important proteins in effecting DNA condensation and reveals that the different associative properties of H-NS and StpA allow them to perform distinct, yet complementary roles in the bacterial nucleoid. more...
- Published
- 2009
- Full Text
- View/download PDF
34. StpA protein from Escherichia coli condenses supercoiled DNA in preference to linear DNA and protects it from digestion by DNase I and EcoKI.
- Author
-
Keatch SA, Leonard PG, Ladbury JE, and Dryden DT
- Subjects
- Adenosine Triphosphate metabolism, DNA, Bacterial chemistry, DNA Restriction Enzymes metabolism, DNA, Bacterial metabolism, DNA, Superhelical metabolism, DNA-Binding Proteins metabolism, Deoxyribonuclease I metabolism, Escherichia coli Proteins metabolism, Molecular Chaperones metabolism
- Abstract
The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250-300 bp of supercoiled DNA and approximately one StpA molecule per 60-100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress. more...
- Published
- 2005
- Full Text
- View/download PDF
35. Apparatus for the micromanipulation of small bacteria.
- Author
-
Isaac L, Ware GC, and Leonard PG
- Subjects
- Bacteria isolation & purification, Bacteriological Techniques instrumentation, Micromanipulation instrumentation
- Published
- 1975
36. Modification of the powell culture chamber for the observation and micromanipulation of growing bacteria.
- Author
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Isaac L, Leonard PG, and Ware GC
- Subjects
- Bacteriological Techniques instrumentation
- Published
- 1975
37. The dental art of ancient Mexico.
- Author
-
Leonard PG
- Subjects
- Art, History of Dentistry, Paleodontology
- Published
- 1972
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