14 results on '"Culhane JC"'
Search Results
2. Real-Time Monitoring of Solid-Liquid Slurries: Optimized Synthesis of Tetrabenazine.
- Author
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Sato Y, Liu J, Kukor AJ, Culhane JC, Tucker JL, Kucera DJ, Cochran BM, and Hein JE
- Subjects
- Chromatography, High Pressure Liquid, Pharmaceutical Preparations, Tetrabenazine
- Abstract
Solid-liquid slurries are vital and increasingly prevalent in the pharmaceutical and chemical industries. Despite the importance of these heterogeneous systems, process control and optimization are fundamentally hindered by a lack of compatible real-time analytical techniques. We present herein an online HPLC monitoring platform enabling access to real-time compositional information on slurries. We demonstrate the system by investigating the heterogeneous synthesis reaction of tetrabenazine. Furthermore, we integrated our online HPLC platform with the orthogonal monitoring techniques of a pH probe and a microscopic imaging probe to provide additional mechanistic insight. These combined insights enable the optimization of tetrabenazine synthesis in terms of reaction time, byproduct formation, and diastereomeric purity of the final product.
- Published
- 2021
- Full Text
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3. An evolved oxazolidinone with selective potency against Mycobacterium tuberculosis and gram positive bacteria.
- Author
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Kaushik A, Heuer AM, Bell DT, Culhane JC, Ebner DC, Parrish N, Ippoliti JT, and Lamichhane G
- Subjects
- Anti-Bacterial Agents chemical synthesis, Anti-Bacterial Agents chemistry, Dose-Response Relationship, Drug, Gram-Negative Bacteria drug effects, Gram-Negative Bacteria growth & development, Gram-Positive Bacteria growth & development, Microbial Sensitivity Tests, Molecular Structure, Oxazolidinones chemical synthesis, Oxazolidinones chemistry, Structure-Activity Relationship, Anti-Bacterial Agents pharmacology, Gram-Positive Bacteria drug effects, Oxazolidinones pharmacology
- Abstract
Innovation of new antibacterials that are effective against strains that have developed resistance to existing drugs would strengthen our ability to treat and subsequently control spread of pathogenic bacteria. Increasing incidence of infections with drug resistant bacteria has become a common occurrence in recent times. We have developed an evolved oxazolidinone, T145, which inhibits growth of Enterococcus faecalis, Staphylococcus aureus and Mycobacterium tuberculosis (Mtb) with sub μg/ml potencies that are potentially therapeutically valuable. The oxazolidinone is bactericidal against Mtb but bacteriostatic against E. faecalis and S. aureus. In addition to therapeutically valuable potency and bactericidal activity against Mtb, T145 minimizes selection of spontaneous resistant mutants, a trait that prolongs longevity of a drug in clinical use., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
4. Structure of Hepatitis C Virus Envelope Glycoprotein E1 Antigenic Site 314-324 in Complex with Antibody IGH526.
- Author
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Kong L, Kadam RU, Giang E, Ruwona TB, Nieusma T, Culhane JC, Stanfield RL, Dawson PE, Wilson IA, and Law M
- Subjects
- Antibodies, Monoclonal immunology, Antibodies, Neutralizing immunology, Binding Sites, Antibody, Cell Line, Crystallography, X-Ray, Epitope Mapping, Epitopes immunology, HEK293 Cells, Hepacivirus immunology, Humans, Molecular Dynamics Simulation, Epitopes ultrastructure, Hepatitis C Antibodies immunology, Immunoglobulin Fab Fragments immunology, Viral Envelope Proteins immunology, Viral Envelope Proteins ultrastructure
- Abstract
Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral "spike" of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
5. Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.
- Author
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Tarrant MK, Rho HS, Xie Z, Jiang YL, Gross C, Culhane JC, Yan G, Qian J, Ichikawa Y, Matsuoka T, Zachara N, Etzkorn FA, Hart GW, Jeong JS, Blackshaw S, Zhu H, and Cole PA
- Subjects
- Animals, Casein Kinase II biosynthesis, Casein Kinase II chemistry, Cell Line, Humans, NIMA-Interacting Peptidylprolyl Isomerase, Peptidylprolyl Isomerase chemistry, Peptidylprolyl Isomerase metabolism, Phosphorylation, Rats, Serine metabolism, Acetylglucosamine metabolism, Casein Kinase II metabolism
- Abstract
Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.
- Published
- 2012
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6. Synthesis and reactivity of sulfamoyl azides and 1-sulfamoyl-1,2,3-triazoles.
- Author
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Culhane JC and Fokin VV
- Subjects
- Azides chemistry, Molecular Structure, Stereoisomerism, Triazoles chemistry, Azides chemical synthesis, Triazoles chemical synthesis
- Abstract
Sulfamoyl azides are readily generated from secondary amines and a novel sulfonyl azide transfer agent, 2,3-dimethyl-1H-imidazolium triflate. They react with alkynes in the presence of a CuTC catalyst forming 1-sulfamoyl-1,2,3-triazoles. The latter are shelf-stable progenitors of rhodium azavinyl carbenes, versatile reactive intermediates that, among other reactions, readily and asymmetrically add to olefins.
- Published
- 2011
- Full Text
- View/download PDF
7. Comparative analysis of small molecules and histone substrate analogues as LSD1 lysine demethylase inhibitors.
- Author
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Culhane JC, Wang D, Yen PM, and Cole PA
- Subjects
- Animals, Cells, Cultured, Enzyme Inhibitors chemical synthesis, Histone Demethylases isolation & purification, Hydrazines chemistry, Pargyline analogs & derivatives, Pargyline chemistry, Peptides chemistry, Propylamines chemistry, Rats, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Structure-Activity Relationship, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Histone Demethylases antagonists & inhibitors, Histones chemistry, Peptides pharmacology
- Abstract
LSD1 is a flavin-dependent histone demethylase that oxidatively removes methyl groups from Lys-4 of histone H3. LSD1 belongs to the amine oxidase enzyme superfamily which utilize molecular oxygen to transform amines to imines that are hydrolytically cleaved to formaldehyde. In prior studies, it has been shown that monoamine oxidase inhibitory scaffolds such as propargylamines and cyclopropylamines can serve as mechanism-based inactivators of LSD1. Propargylamine-histone H3 peptide analogues are potent LSD1 inhibitors, whereas small molecule antidepressant MAO acetylenic inhibitors like pargyline do not inhibit LSD1. In contrast, the small molecule MAO cyclopropylamine inhibitor tranylcypromine is a time-dependent LSD1 inhibitor but exo-cyclopropylamine-peptide substrate analogue is not. To provide further insight into small molecule versus peptide relationships in LSD1 inhibition, herein we further our analysis of warheads in peptide scaffolds to include the chlorovinyl, endo-cyclopropylamine, and hydrazine-functionalities as LSD1 inactivators. We find that chlorovinyl-H3 is a mechanism-based LSD1 inactivator whereas endo-cyclopropylamine-H3 does not show time-dependent inactivation. The hydrazine-H3 was shown to be the most potent LSD1 suicide inhibitor yet reported, more than 20-fold more efficient in inhibiting demethylation than propargylamine-H3 derivatives. We re-explored MAO antidepressant agent phenelzine (phenethylhydrazine), previously reported to be a weak LSD1 inhibitor, and found that it is far more potent than previously appreciated. We show that phenelzine can block histone H3K4Me demethylation in cells, validating it as a pharmacologic tool and potential lead structure for anticancer therapy.
- Published
- 2010
- Full Text
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8. Synthesis of novel oxazolidinone antimicrobial agents.
- Author
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Ebner DC, Culhane JC, Winkelman TN, Haustein MD, Ditty JL, and Ippoliti JT
- Subjects
- Anti-Bacterial Agents chemistry, Escherichia coli drug effects, Molecular Structure, Oxazolidinones chemistry, Staphylococcus aureus drug effects, Structure-Activity Relationship, Anti-Bacterial Agents chemical synthesis, Anti-Bacterial Agents pharmacology, Oxazolidinones chemical synthesis, Oxazolidinones pharmacology
- Abstract
The oxazolidinone class of antimicrobials represents a promising advance in the fight against resistant Gram-positive bacterial infections. Four novel oxazolidinone antimicrobial compounds, each containing a benzodioxin ring system, have been prepared. The general synthesis of each compound begins with the construction of a benzodioxin ring system containing a nitro substituent that ultimately becomes the nitrogen of the oxazolidinone ring. Three of the compounds utilize high yielding 'click chemistry' in their final step. The antimicrobial activities of the new oxazolidinones have been measured and the MIC against Staphylococcus aureus for one of the antimicrobials was determined to be 2-3 microg/mL, which is comparable to the well-known oxazolidinone, linezolid.
- Published
- 2008
- Full Text
- View/download PDF
9. De novo discovery of serotonin N-acetyltransferase inhibitors.
- Author
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Szewczuk LM, Saldanha SA, Ganguly S, Bowers EM, Javoroncov M, Karanam B, Culhane JC, Holbert MA, Klein DC, Abagyan R, and Cole PA
- Subjects
- Acetyl Coenzyme A antagonists & inhibitors, Acetyl Coenzyme A chemistry, Animals, Arylalkylamine N-Acetyltransferase biosynthesis, Binding Sites, Cells, Cultured, Crystallography, X-Ray, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, Melatonin antagonists & inhibitors, Melatonin biosynthesis, Pineal Gland cytology, Protein Binding, Protein Conformation, Rats, Rhodanine analogs & derivatives, Rhodanine chemistry, Rhodanine pharmacology, Tryptamines chemistry, Tryptamines pharmacology, Arylalkylamine N-Acetyltransferase antagonists & inhibitors, Arylalkylamine N-Acetyltransferase chemistry, Enzyme Inhibitors chemistry, Models, Molecular, Quantitative Structure-Activity Relationship
- Abstract
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is a member of the GCN5 N-acetyltransferase (GNAT) superfamily and catalyzes the penultimate step in the biosynthesis of melatonin; a large daily rhythm in AANAT activity drives the daily rhythm in circulating melatonin. We have used a structure-based computational approach to identify the first druglike and selective inhibitors of AANAT. Approximately 1.2 million compounds were virtually screened by 3D high-throughput docking into the active site of X-ray structures for AANAT, and in total 241 compounds were tested as inhibitors. One compound class, containing a rhodanine scaffold, exhibited low micromolar competitive inhibition against acetyl-CoA (AcCoA) and proved to be effective in blocking melatonin production in pineal cells. Compounds from this class are predicted to bind as bisubstrate inhibitors through interactions with the AcCoA and serotonin binding sites. Overall, this study demonstrates the feasibility of using virtual screening to identify small molecules that are selective inhibitors of AANAT.
- Published
- 2007
- Full Text
- View/download PDF
10. LSD1 and the chemistry of histone demethylation.
- Author
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Culhane JC and Cole PA
- Subjects
- Catalysis, Enzyme Inhibitors, F-Box Proteins, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Methylation, Substrate Specificity, Chromatin Assembly and Disassembly, Histones metabolism, Oxidoreductases, N-Demethylating metabolism, Protein Processing, Post-Translational
- Abstract
The recent discovery that histone demethylation can be catalyzed by the flavin-dependent amine oxidase LSD1 has ushered in a new chapter in the chromatin-remodeling community. Herein, we discuss the rapid progress of the histone demethylase field including the recent identification of the non-heme iron-dependent histone demethylases (JmjC family), the basis for LSD1 substrate site specificity and the newly emerging potential for inhibition of these enzymes in structural and functional analysis.
- Published
- 2007
- Full Text
- View/download PDF
11. Structural basis for the inhibition of the LSD1 histone demethylase by the antidepressant trans-2-phenylcyclopropylamine.
- Author
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Yang M, Culhane JC, Szewczuk LM, Jalili P, Ball HL, Machius M, Cole PA, and Yu H
- Subjects
- Histone Demethylases, Kinetics, Models, Molecular, Oxidoreductases, N-Demethylating chemistry, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Spectrophotometry, Ultraviolet, Antidepressive Agents pharmacology, Enzyme Inhibitors pharmacology, Oxidoreductases, N-Demethylating antagonists & inhibitors, Tranylcypromine pharmacology
- Abstract
Histone modifications, such as acetylation and methylation, are important epigenetic marks that regulate diverse biological processes that use chromatin as the template, including transcription. Dysregulation of histone acetylation and methylation leads to the silencing of tumor suppressor genes and contributes to cancer progression. Inhibitors of enzymes that catalyze the addition and removal of these epigenetic marks thus have therapeutic potential for treating cancer. Lysine-specific demethylase 1 (LSD1) is the first discovered histone lysine demethylase and, with the help of its cofactor CoREST, specifically demethylates mono- and dimethylated histone H3 lysine 4 (H3-K4), thus repressing transcription. Because LSD1 belongs to the family of flavin adenine dinucleotide (FAD)-dependent amine oxidases, certain inhibitors of monoamine oxidases (MAOs), including the clinically used antidepressant trans-2-phenylcyclopropylamine (PCPA; tranylcypromine; Parnate), are also capable of inhibiting LSD1. In this study, we have further measured the kinetic parameters of the inhibition of LSD1 by PCPA and determined the crystal structure of LSD1-CoREST in the presence of PCPA. Our structural and mass spectrometry analyses are consistent with PCPA forming a covalent adduct with FAD in LSD1 that is distinct from the FAD-PCPA adduct of MAO B. The structure also reveals that the phenyl ring of the FAD-PCPA adduct in LSD1 does not form extensive interactions with active-site residues. This study thus provides the basis for designing more potent inhibitors of LSD1 that contain substitutions on the phenyl ring of PCPA to fully engage neighboring residues.
- Published
- 2007
- Full Text
- View/download PDF
12. Mechanistic analysis of a suicide inactivator of histone demethylase LSD1.
- Author
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Szewczuk LM, Culhane JC, Yang M, Majumdar A, Yu H, and Cole PA
- Subjects
- Amino Acid Sequence, Biotinylation, Enzyme Inhibitors chemistry, Flavins pharmacology, Glutathione metabolism, HeLa Cells, Histone Demethylases, Humans, Kinetics, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Oxidation-Reduction, Oxidoreductases, N-Demethylating isolation & purification, Spectrophotometry, Enzyme Inhibitors pharmacology, Oxidoreductases, N-Demethylating antagonists & inhibitors, Oxidoreductases, N-Demethylating chemistry
- Abstract
Lysine-specific demethylase 1 (LSD1) is a transcriptional repressor and a flavin-dependent amine oxidase that is responsible for the removal of methyl from lysine 4 of histone H3. In this study, we characterize the mechanism and scope of LSD1 inhibition by a propargylamine-derivatized histone H3 substrate (1). Unlike aziridinyl and cyclopropylamine-derivatized histone H3 peptide substrate analogues, compound 1 appears to covalently modify and irreversibly inactivate LSD1 with high potency. Accompanying this inactivation is a spectroscopic change, which shifts the absorbance maximum to 392 nm. Spectral changes associated with the 1-LSD1 complex and reactivity to decreased pH and sodium borohydride treatment were suggestive of a structure involving a flavin-linked inhibitor conjugate between N5 of the flavin and the terminal carbon of the inhibitor. Using a 13C-labeled inhibitor, NMR analysis of the 1-flavin conjugate was consistent with this structural assignment. Kinetic analysis of the spectroscopic shift induced by 1 showed that the flavin adduct formed in a reaction with kinetic constants similar to those of the LSD1 inactivation process. Taken together, these data support a mechanism of LSD1 inactivation by 1 involving amine oxidation followed by Michael addition to the propargylic imine. We further examined the potential for a biotinylated analogue of 1 (1-Btn) to be used as a tool in affinity pulldown experiments. Using 1-Btn, it was feasible to selectively pull down spiked and endogenous LSD1 from HeLa cell nuclear extracts, setting the stage for activity-based demethylase proteomics.
- Published
- 2007
- Full Text
- View/download PDF
13. Structural basis of histone demethylation by LSD1 revealed by suicide inactivation.
- Author
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Yang M, Culhane JC, Szewczuk LM, Gocke CB, Brautigam CA, Tomchick DR, Machius M, Cole PA, and Yu H
- Subjects
- Amino Acid Sequence, Crystallization, Histone Demethylases, Histones genetics, Humans, Methylation, Molecular Sequence Data, Molecular Structure, Mutagenesis, Substrate Specificity, Gene Silencing physiology, Histones metabolism, Models, Molecular, Oxidoreductases, N-Demethylating genetics
- Abstract
Histone methylation regulates diverse chromatin-templated processes, including transcription. The recent discovery of the first histone lysine-specific demethylase (LSD1) has changed the long-held view that histone methylation is a permanent epigenetic mark. LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that demethylates histone H3 Lys4 (H3-K4). However, the mechanism by which LSD1 achieves its substrate specificity is unclear. We report the crystal structure of human LSD1 with a propargylamine-derivatized H3 peptide covalently tethered to FAD. H3 adopts three consecutive gamma-turns, enabling an ideal side chain spacing that places its N terminus into an anionic pocket and positions methyl-Lys4 near FAD for catalysis. The LSD1 active site cannot productively accommodate more than three residues on the N-terminal side of the methyllysine, explaining its H3-K4 specificity. The unusual backbone conformation of LSD1-bound H3 suggests a strategy for designing potent LSD1 inhibitors with therapeutic potential.
- Published
- 2007
- Full Text
- View/download PDF
14. A mechanism-based inactivator for histone demethylase LSD1.
- Author
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Culhane JC, Szewczuk LM, Liu X, Da G, Marmorstein R, and Cole PA
- Subjects
- Amino Acid Sequence, Enzyme Activation, Flavin-Adenine Dinucleotide analogs & derivatives, Flavin-Adenine Dinucleotide chemistry, Flavin-Adenine Dinucleotide metabolism, Histone Deacetylases metabolism, Kinetics, Lysine chemistry, Lysine pharmacology, Molecular Sequence Data, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Histone Deacetylase Inhibitors, Histone Deacetylases chemistry, Lysine analogs & derivatives
- Abstract
Histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the oxidative removal of one or two methyl groups from the methyl-lysine-4 side chain of histone H3. We have designed and synthesized two peptide-based inhibitor analogues that block LSD1. One of these inhibitors, compound 1, contains a propargylamine functionality and shows time-dependent inactivation of LSD1. Peptide substrate, diMeK4H3-21, protected LSD1 against inactivation by 1 in a concentration-dependent fashion. Mass spectrometric analysis showed that 1 forms a covalent interaction with FAD. Compound 1 did not detectably inhibit monoamine oxidase B in the concentration range studied. Compound 1 is thus a selective, mechanism-based inactivator of LSD1 and is likely to serve as a useful tool in the study of histone modifications and chromatin remodeling.
- Published
- 2006
- Full Text
- View/download PDF
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