114 results on '"John B. Bruning"'
Search Results
2. Engineering C–C Bond Cleavage Activity into a P450 Monooxygenase Enzyme
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Justin C. Miller, Joel H. Z. Lee, Mark A. Mclean, Rebecca R. Chao, Isobella S. J. Stone, Tara L. Pukala, John B. Bruning, James J. De Voss, Mary A. Schuler, Stephen G. Sligar, and Stephen G. Bell
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Colloid and Surface Chemistry ,General Chemistry ,Biochemistry ,Catalysis - Published
- 2023
3. Escherichia coli YgiC and YjfC Possess Peptide─Spermidine Ligase Activity
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Jordan L. Pederick, Jack Klose, Blagojce Jovcevski, Tara L. Pukala, and John B. Bruning
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Biochemistry - Published
- 2023
4. A New 1,2,3-Triazole Scaffold with Improved Potency against Staphylococcus aureus Biotin Protein Ligase
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Damian L. Stachura, Stephanie Nguyen, Steven W. Polyak, Blagojce Jovcevski, John B. Bruning, Andrew D. Abell, Stachura, Damian L, Nguyen, Stephanie, Polyak, Steven W, Jovcevski, Blagojce, Bruning, John B, and Abell, Andrew D
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Staphylococcus aureus ,triazole ,Infectious Diseases ,antibiotic ,biotin protein ligase - Abstract
Refereed/Peer-reviewed Staphylococcus aureus, a key ESKAPE bacteria, is responsible for most blood-based infections and, as a result, is a major economic healthcare burden requiring urgent attention. Here, we report in silico docking, synthesis, and assay of N1-diphenylmethyl triazole-based analogues (7–13) designed to interact with the entire binding site of S. aureus biotin protein ligase (SaBPL), an enzyme critical for the regulation of gluconeogenesis and fatty acid biosynthesis. The second aryl ring of these compounds enhances both SaBPL potency and whole cell activity against S. aureus relative to previously reported mono-benzyl triazoles. Analogues 12 and 13, with added substituents to better interact with the adenine binding site, are particularly potent, with Ki values of 6.01 ± 1.01 and 8.43 ± 0.73 nM, respectively. These analogues are the most active triazole-based inhibitors reported to date and, importantly, inhibit the growth of a clinical isolate strain of S. aureus ATCC 49775, with minimum inhibitory concentrations of 1 and 8 μg/mL, respectively.
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- 2022
5. The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum
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Amna Ghith, Daniel Z. Doherty, John B. Bruning, Robert A. Russell, James J. De Voss, and Stephen G. Bell
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Infectious Diseases - Published
- 2022
6. Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase
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Wanita Pantong, Jordan L. Pederick, Somchart Maenpuen, Ruchanok Tinikul, Jaime J. Jayapalan, Blagojce Jovcevski, Kate L. Wegener, John B. Bruning, and Wanisa Salaemae
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Molecular Biology ,Biochemistry - Published
- 2023
7. Identification and characterisation of MdUGT78T2 as a galactosyltransferase with dual activity on flavonol and anthocyanidin substrates in red-skinned apple fruit (Malus domestica L.)
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Daniel Clayton-Cuch, Daniel McDougal, Julian G. Schwerdt, Long Yu, Neil Shirley, David Bradley, John B. Bruning, Christine Böttcher, and Vincent Bulone
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General Medicine ,Food Science ,Analytical Chemistry - Published
- 2023
8. Fortuitousin vitrocompound degradation produces a tractable hit againstMycobacterium tuberculosisdethiobiotin synthetase: a cautionary tale of what goes in, does not always come out
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Wanisa Salaemae, Andrew P. Thompson, Birgit I. Gaiser, Kwang Jun Lee, Michael T. Huxley, Christopher J. Sumby, Steven W. Polyak, Andrew D. Abell, John B. Bruning, and Kate L. Wegener
- Abstract
We previously reported potent ligands and inhibitors ofMycobacterium tuberculosisdethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumannet al.,ACS Chem Biol. 2021, 16, 2339-2347); here the unconventional origin of the fragment compound they were derived from is described for the first time. Compound1(9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified byin silicofragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket, when1was soaked intoMtDTBS crystals, but the density was inconsistent with the structure of1. Here we show the lactone of1hydrolyses to carboxylic acid2under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid3. Crystals soaked directly with authentic3produced electron density that matched that of crystals soaked with presumed1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed3enabled subsequent compound development into nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process.SynopsisA molecule flagged in anin silicodocking screen againstMtDTBS, was inadvertently hydrolysed in the crystal conditions used for hit validation. The resulting fragment-sized molecule bound to the DAPA substrate binding pocket of the target enzyme (MtDTBS) with millimolar affinity, as measured by surface plasmon resonance, but was later modified to a highly potent (nanomolar) ligand and promising lead for the development of novel tuberculosis treatments.Graphical Abstract
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- 2023
9. Cytochrome P450-Catalyzed Oxidation of Halogen-Containing Substrates
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Tom Coleman, Matthew N. Podgorski, Maya L. Doyle, Jarred M. Scaffidi-Muta, Eleanor C. Campbell, John B. Bruning, James J. De Voss, and Stephen G. Bell
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Inorganic Chemistry ,History ,Polymers and Plastics ,Business and International Management ,Biochemistry ,Industrial and Manufacturing Engineering - Published
- 2023
10. Structural study of potent triazole-based inhibitors of Staphylococcus aureus biotin protein ligase
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Damian L. Stachura, Stephanie Nguyen, Steven W. Polyak, Blagojce Jovcevski, John B. Bruning, Andrew D. Abell, Stachura, Damian L, Nguyen, Stephanie, Polyak, Steven W, Jovcevski, Blagojce, Bruning, John B, and Abell, Andrew D
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inhibitor ,Staphylococcus aureus ,triazole ,antibiotic ,Organic Chemistry ,Drug Discovery ,biotin protein ligase ,Biochemistry - Abstract
The rise of multidrug-resistant bacteria, such as Staphylococcus aureus, has highlighted global urgency for new classes of antibiotics. Biotin protein ligase (BPL), a critical metabolic regulatory enzyme, is an important target that shows significant promise in this context. Here we report the in silico docking, synthesis, and biological assay of a new series of N1- diphenylmethyl-1,2,3-triazole-based S. aureus BPL (SaBPL) inhibitors (8-19) designed to probe the adenine binding site and define whole-cell activity for this important class of inhibitor. Triazoles 13 and 14 with N1-propylamine and-butanamide substituents, respectively, were particularly potent with Ki values of 10 +/- 2 and 30 +/- 6 nM, respectively, against SaBPL. A strong correlation was apparent between the Ki values for 8-19 and the in silico docking, with hydrogen bonding to amino acid residues S128 and N212 of SaBPL likely contributing to potent inhibition. Refereed/Peer-reviewed
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- 2023
11. A structural model of the human plasminogen and Aspergillus fumigatus enolase complex
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Stephanie Nguyen, Blagojce Jovcevski, Jia Q. Truong, Tara L. Pukala, and John B. Bruning
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Models, Structural ,Phosphoenolpyruvate ,Antifungal Agents ,Structural Biology ,Aspergillus fumigatus ,Phosphopyruvate Hydratase ,Humans ,Plasminogen ,Molecular Biology ,Biochemistry ,Protein Binding - Abstract
The metabolic enzyme, enolase, plays a crucial role in the cytoplasm where it maintains cellular energy production within the process of glycolysis. The main role of enolase in glycolysis is to convert 2-phosphoglycerate to phosphoenolpyruvate; however, enolase can fulfill roles that deviate from this function. In pathogenic bacteria and fungi, enolase is also located on the cell surface where it functions as a virulence factor. Surface-expressed enolase is a receptor for human plasma proteins, including plasminogen, and this interaction facilitates nutrient acquisition and tissue invasion. A novel approach to developing antifungal drugs is to inhibit the formation of this complex. To better understand the structure of enolase and the interactions that may govern complex formation, we have solved the first X-ray crystal structure of enolase from Aspergillus fumigatus (2.0 Å) and have shown that it preferentially adopts a dimeric quaternary structure using native mass spectrometry. Two additional X-ray crystal structures of A. fumigatus enolase bound to the endogenous substrate 2-phosphoglycerate and product phosphoenolpyruvate were determined and kinetic characterization was carried out to better understand the details of its canonical function. From these data, we have produced a model of the A. fumigatus enolase and human plasminogen complex to provide structural insights into the mechanisms of virulence and aid future development of small molecules or peptidomimetics for antifungal drug design.
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- 2022
12. An Altered Heme Environment in an Engineered Cytochrome P450 Enzyme Enables the Switch from Monooxygenase to Peroxygenase Activity
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Matthew N. Podgorski, Joshua S. Harbort, Joel H. Z. Lee, Giang T.H. Nguyen, John B. Bruning, William A. Donald, Paul V. Bernhardt, Jeffrey R. Harmer, and Stephen G. Bell
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General Chemistry ,Catalysis - Published
- 2022
13. Different Geometric Requirements for Cytochrome P450-Catalyzed Aliphatic Versus Aromatic Hydroxylation Results in Chemoselective Oxidation
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Tom Coleman, Alicia M. Kirk, Joel H. Z. Lee, Daniel Z. Doherty, John B. Bruning, Elizabeth H. Krenske, James J. De Voss, and Stephen G. Bell
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General Chemistry ,Catalysis - Published
- 2022
14. The oxidation of cholesterol derivatives by the CYP124 and CYP142 enzymes from Mycobacterium marinum
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Amna Ghith, John B. Bruning, and Stephen G. Bell
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Endocrinology ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Molecular Medicine ,Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2023
15. A New 1,2,3-Triazole Scaffold with Improved Potency against
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Damian L, Stachura, Stephanie, Nguyen, Steven W, Polyak, Blagojce, Jovcevski, John B, Bruning, and Andrew D, Abell
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- 2022
16. Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation
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Tom Coleman, Daniel Z. Doherty, Ting Zhang, Matthew N. Podgorski, Ruihong Qiao, Joel H. Z. Lee, John B. Bruning, James J. De Voss, Weihong Zhou, and Stephen G. Bell
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Organic Chemistry ,General Chemistry ,Biochemistry - Abstract
The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon-hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4-ethyl- and 4-isopropyl- benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4-cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4-n-propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4-n-butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4-isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4-n-propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X-ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4-n-propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.
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- 2022
17. Investigating the Active Oxidants Involved in Cytochrome P450 Catalyzed Sulfoxidation Reactions
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Matthew N. Podgorski, Tom Coleman, Luke R. Churchman, John B. Bruning, James J. De Voss, and Stephen G. Bell
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Organic Chemistry ,General Chemistry ,Catalysis - Abstract
Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C-H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H
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- 2022
18. Inhibition of Mycobacterium tuberculosis Dethiobiotin Synthase (MtDTBS): Toward Next-Generation Antituberculosis Agents
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Jordan L. Pederick, Birgit I Gaiser, Nicholas C Schumann, Kate L. Wegener, Kwangjun Lee, James Hodgkinson-Bean, Steven W. Polyak, Thomas D. Avery, Grant W. Booker, John B. Bruning, Andrew D. Abell, Wanisa Salaemae, and Andrew P. Thompson
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Tuberculosis ,In silico ,01 natural sciences ,Biochemistry ,Dethiobiotin synthase ,Mycobacterium tuberculosis ,03 medical and health sciences ,chemistry.chemical_compound ,Biotin ,Biosynthesis ,medicine ,Tetrazole ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,010405 organic chemistry ,General Medicine ,medicine.disease ,biology.organism_classification ,0104 chemical sciences ,3. Good health ,Enzyme ,chemistry ,biology.protein ,Molecular Medicine - Abstract
Mycobacterium tuberculosis dethiobiotin synthase (MtDTBS) is a crucial enzyme involved in the biosynthesis of biotin in the causative agent of tuberculosis, M. tuberculosis. Here, we report a binder of MtDTBS, cyclopentylacetic acid 2 (KD = 3.4 ± 0.4 mM), identified via in silico screening. X-ray crystallography showed that 2 binds in the 7,8-diaminopelargonic acid (DAPA) pocket of MtDTBS. Appending an acidic group to the para-position of the aromatic ring of the scaffold revealed compounds 4c and 4d as more potent binders, with KD = 19 ± 5 and 17 ± 1 μM, respectively. Further optimization identified tetrazole 7a as a particularly potent binder (KD = 57 ± 5 nM) and inhibitor (Ki = 5 ± 1 μM) of MtDTBS. Our findings highlight the first reported inhibitors of MtDTBS and serve as a platform for the further development of potent inhibitors and novel therapeutics for the treatment of tuberculosis.
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- 2021
19. The Stereoselective Oxidation of para ‐Substituted Benzenes by a Cytochrome P450 Biocatalyst
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Ian C-K Lau, Joel H. Z. Lee, John B. Bruning, James J. De Voss, Rebecca R. Chao, Stephen Bell, T. Coleman, Stella A. Child, and Luke R. Churchman
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biology ,010405 organic chemistry ,Stereochemistry ,Organic Chemistry ,Active site ,Substrate (chemistry) ,Benzene ,General Chemistry ,Monooxygenase ,Hydroxylation ,010402 general chemistry ,01 natural sciences ,Catalysis ,Substrate Specificity ,0104 chemical sciences ,chemistry.chemical_compound ,Cytochrome P-450 Enzyme System ,chemistry ,biology.protein ,Moiety ,Stereoselectivity ,Carboxylate ,Oxidation-Reduction ,Benzoic acid - Abstract
The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP).min) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.
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- 2021
20. Engineering potassium activation into biosynthetic thiolase
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John B. Bruning and Andrew C Marshall
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Models, Molecular ,Protein Conformation ,Stereochemistry ,Allosteric regulation ,Crystallography, X-Ray ,Protein Engineering ,Biochemistry ,Substrate Specificity ,Conserved sequence ,Residue (chemistry) ,Enzyme activator ,Bacterial Proteins ,Acetyl Coenzyme A ,Acetyl-CoA C-Acetyltransferase ,Binding site ,Molecular Biology ,Zoogloea ,Thiolase ,Substrate (chemistry) ,Cell Biology ,Protein engineering ,Cations, Monovalent ,Enzyme Activation ,Kinetics ,Mutation ,Biocatalysis ,Potassium ,Acyl Coenzyme A ,Protein Multimerization ,Protein Binding - Abstract
Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation–π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation–π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.
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- 2021
21. Constitutive JAK/STAT signaling is the primary mechanism of resistance to JAKi in TYK2-rearranged acute lymphoblastic leukemia
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Paniz Tavakoli Shirazi, Elyse C. Page, Susan L. Heatley, Deborah L. White, Laura N Eadie, and John B. Bruning
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0301 basic medicine ,Drug ,Cancer Research ,medicine.drug_class ,Lymphoblastic Leukemia ,media_common.quotation_subject ,Proto-Oncogene Proteins c-myb ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Janus Kinase Inhibitors ,Medicine ,Sulfones ,media_common ,Gene Rearrangement ,TYK2 Kinase ,Oncogene ,Mechanism (biology) ,business.industry ,Histone deacetylase inhibitor ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,Fusion protein ,In vitro ,STAT Transcription Factors ,Pyrimidines ,030104 developmental biology ,Oncology ,Drug Resistance, Neoplasm ,Tyrosine kinase 2 ,030220 oncology & carcinogenesis ,Cancer research ,business ,Signal Transduction - Abstract
Activating TYK2-rearrangements have recently been identified and implicated in the leukemogenesis of high-risk acute lymphoblastic leukemia (HR-ALL) cases. Pre-clinical studies indicated the JAK/TYK2 inhibitor (JAKi), cerdulatinib, as a promising therapeutic against TYK2-rearranged ALL, attenuating the constitutive JAK/STAT signaling resulting from the TYK2 fusion protein. However, following a period of clinical efficacy, JAKi resistance often occurs resulting in relapse. In this study, we modeled potential mechanisms of JAKi resistance in TYK2-rearranged ALL cells in vitro in order to recapitulate possible clinical scenarios and provide a rationale for alternative therapies. Cerdulatinib resistant B-cells, driven by the MYB-TYK2 fusion oncogene, were generated by long-term exposure to the drug. Sustained treatment of MYB-TYK2-rearranged ALL cells with cerdulatinib led to enhanced and persistent JAK/STAT signaling, co-occurring with JAK1 overexpression. Hyperactivation of JAK/STAT signaling and JAK1 overexpression was reversible as cerdulatinib withdrawal resulted in re-sensitization to the drug. Importantly, histone deacetylase inhibitor (HDACi) therapies were efficacious against cerdulatinib-resistant cells demonstrating a potential alternative therapy for use in TYK2-rearranged B-ALL patients who have lost response to JAKi treatment regimens.
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- 2021
22. Acquired JAK2 mutations confer resistance to JAK inhibitors in cell models of acute lymphoblastic leukemia
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Charlotte E J Downes, James Breen, David T Yeung, Deborah L. White, Barbara J. McClure, John B. Bruning, Jacqueline Rehn, and Elyse C. Page
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Drug ,Cancer Research ,Mutation ,Ruxolitinib ,Acute lymphocytic leukaemia ,Mechanism (biology) ,business.industry ,media_common.quotation_subject ,Lymphoblastic Leukemia ,Phases of clinical research ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,medicine.disease_cause ,In vitro ,Article ,Cancer therapeutic resistance ,Targeted therapies ,Oncology ,hemic and lymphatic diseases ,medicine ,Cancer research ,Binding site ,business ,RC254-282 ,media_common ,medicine.drug - Abstract
Ruxolitinib (rux) Phase II clinical trials are underway for the treatment of high-riskJAK2-rearranged (JAK2r) B-cell acute lymphoblastic leukemia (B-ALL). Treatment resistance to targeted inhibitors in other settings is common; elucidating potential mechanisms of rux resistance inJAK2r B-ALL will enable development of therapeutic strategies to overcome or avert resistance. We generated a murine pro-B cell model ofATF7IP-JAK2with acquired resistance to multiple type-I JAK inhibitors. Resistance was associated with mutations within theJAK2ATP/rux binding site, including aJAK2p.G993A mutation. Using in vitro models ofJAK2r B-ALL,JAK2p.G993A conferred resistance to six type-I JAK inhibitors and the type-II JAK inhibitor, CHZ-868. Using computational modeling, we postulate thatJAK2p.G993A enabled JAK2 activation in the presence of drug binding through a unique resistance mechanism that modulates the mobility of the conserved JAK2 activation loop. This study highlights the importance of monitoring mutation emergence and may inform future drug design and the development of therapeutic strategies for this high-risk patient cohort.
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- 2021
23. Immunogenicity study of engineered ferritins with C- and N-terminus insertion of Epstein-Barr nuclear antigen 1 epitope
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Shuang Yin, Jordan L. Pederick, Yan Sun, Yiran Qu, Yingli Wang, Bingyang Zhang, John B. Bruning, Jingxiu Bi, and Anton P. J. Middelberg
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Epstein-Barr Virus Infections ,Herpesvirus 4, Human ,General Veterinary ,General Immunology and Microbiology ,biology ,Chemistry ,Immunogenicity ,Public Health, Environmental and Occupational Health ,Antibody titer ,Molecular biology ,Epitope ,Ferritin ,Epitopes ,Infectious Diseases ,Immune system ,Epstein-Barr Virus Nuclear Antigens ,Antigen ,Ferritins ,Splenocyte ,biology.protein ,Humans ,Molecular Medicine ,Immunogenicity Study - Abstract
Human ferritin heavy chain, an example of a protein nanoparticle, has recently been used as a vaccine delivery platform. Human ferritin has advantages of uniform architecture, robust thermal and chemical stabilities, and good biocompatibility and biodegradation. There is however a lack of understanding about the relationship between insertion sites in ferritin (N-terminus and C-terminus) and the corresponding humoral and cell-mediated immune responses. To bridge this gap, we utilized an Epstein-Barr Nuclear Antigen 1 (EBNA1) epitope as a model to produce engineered ferritin-based vaccines E1F1 (N-terminus insertion) and F1E1 (C-terminus insertion) for the prevention of Epstein-Barr virus (EBV) infections. X-ray crystallography confirmed the relative positions of the N-terminus insertion and C-terminus insertion. For N-terminus insertion, the epitopes were located on the exterior surface of ferritin, while for C-terminus insertion, the epitopes were inside the ferritin cage. Based on the results of antigen-specific antibody titers from in-vivo tests, we found that there was no obvious difference on humoral immune responses between N-terminus and C-terminus insertion. We also evaluated splenocyte proliferation and memory lymphocyte T cell differentiation. Both results suggested C-terminus insertion produced a stronger proliferative response and cell-mediated immune response than N-terminus insertion. C-terminus insertion of EBNA1 epitope was also processed more efficiently by dendritic cells (DCs) than N-terminus insertion. This provides new insight into the relationship between the insertion site and immunogenicity of ferritin nanoparticle vaccines.
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- 2021
24. The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from
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Amna, Ghith, Daniel Z, Doherty, John B, Bruning, Robert A, Russell, James J, De Voss, and Stephen G, Bell
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Cholesterol ,Cytochrome P-450 Enzyme System ,Mycobacterium ulcerans ,Mycobacterium marinum ,Humans ,Tuberculosis ,Mycobacterium tuberculosis - Abstract
The steroid binding CYP142 cytochrome P450 enzymes of
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- 2022
25. JAK2 Alterations in Acute Lymphoblastic Leukemia: Molecular Insights for Superior Precision Medicine Strategies
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Charlotte EJ. Downes, Barbara J. McClure, Daniel P. McDougal, Susan L. Heatley, John B. Bruning, Daniel Thomas, David T. Yeung, and Deborah L. White
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Cell Biology ,Developmental Biology - Abstract
Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, arising from immature lymphocytes that show uncontrolled proliferation and arrested differentiation. Genomic alterations affecting Janus kinase 2 (JAK2) correlate with some of the poorest outcomes within the Philadelphia-like subtype of ALL. Given the success of kinase inhibitors in the treatment of chronic myeloid leukemia, the discovery of activating JAK2 point mutations and JAK2 fusion genes in ALL, was a breakthrough for potential targeted therapies. However, the molecular mechanisms by which these alterations activate JAK2 and promote downstream signaling is poorly understood. Furthermore, as clinical data regarding the limitations of approved JAK inhibitors in myeloproliferative disorders matures, there is a growing awareness of the need for alternative precision medicine approaches for specific JAK2 lesions. This review focuses on the molecular mechanisms behind ALL-associated JAK2 mutations and JAK2 fusion genes, known and potential causes of JAK-inhibitor resistance, and how JAK2 alterations could be targeted using alternative and novel rationally designed therapies to guide precision medicine approaches for these high-risk subtypes of ALL.
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- 2022
26. TSC-insensitive Rheb mutations induce oncogenic transformation through a combination of constitutively active mTORC1 signalling and proteome remodelling
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John B. Bruning, Timothy J. Sargeant, Sean J. Humphrey, Jianling Xie, Wenru Pan, Leanne K. Hein, Christopher G. Proud, Luke A. Selth, and Stuart P. De Poi
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Pharmacology ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Autophagy ,Cell Biology ,mTORC1 ,EEF2 ,Cell biology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Anaerobic glycolysis ,biology.protein ,Molecular Medicine ,Translation factor ,Protein kinase A ,Molecular Biology ,PI3K/AKT/mTOR pathway ,RHEB - Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5' AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.
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- 2021
27. Discovery of an ʟ-amino acid ligase implicated in Staphylococcal sulfur amino acid metabolism
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Jordan L. Pederick, Aimee J. Horsfall, Blagojce Jovcevski, Jack Klose, Andrew D. Abell, Tara L. Pukala, and John B. Bruning
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Aspartic Acid ,Staphylococcus aureus ,Adenosine Triphosphate ,Methionine ,Bacterial Proteins ,Cell Biology ,Dipeptides ,Cysteine ,Peptide Synthases ,Molecular Biology ,Biochemistry ,Phylogeny - Abstract
Enzymes involved in Staphylococcus aureus amino acid metabolism have recently gained traction as promising targets for the development of new antibiotics, however, not all aspects of this process are understood. The ATP-grasp superfamily includes enzymes that predominantly catalyze the ATP-dependent ligation of various carboxylate and amine substrates. One subset, ʟ-amino acid ligases (LALs), primarily catalyze the formation of dipeptide products in Gram-positive bacteria, however, their involvement in S. aureus amino acid metabolism has not been investigated. Here, we present the characterization of the putative ATP-grasp enzyme (SAOUHSC_02373) from S. aureus NCTC 8325 and its identification as a novel LAL. First, we interrogated the activity of SAOUHSC_02373 against a panel of ʟ-amino acid substrates. As a result, we identified SAOUHSC_02373 as an LAL with high selectivity for ʟ-aspartate and ʟ-methionine substrates, specifically forming an ʟ-aspartyl-ʟ-methionine dipeptide. Thus, we propose that SAOUHSC_02373 be assigned as ʟ-aspartate-ʟ-methionine ligase (LdmS). To further understand this unique activity, we investigated the mechanism of LdmS by X-ray crystallography, molecular modeling, and site-directed mutagenesis. Our results suggest that LdmS shares a similar mechanism to other ATP-grasp enzymes but possesses a distinctive active site architecture that confers selectivity for the ʟ-Asp and ʟ-Met substrates. Phylogenetic analysis revealed LdmS homologs are highly conserved in Staphylococcus and closely related Gram-positive Firmicutes. Subsequent genetic analysis upstream of the ldmS operon revealed several trans-acting regulatory elements associated with control of Met and Cys metabolism. Together, these findings support a role for LdmS in Staphylococcal sulfur amino acid metabolism.
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- 2022
28. Vanishing white matter
- Author
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Eline M.C. Hamilton, Stephanie Nguyen, Chris de Graaf, Iwan J. P. de Esch, Christopher G. Proud, John B. Bruning, Lisanne E. Wisse, Marjo S. van der Knaap, Inna Slynko, Truus Em Abbink, Medicinal chemistry, Chemistry and Pharmaceutical Sciences, AIMMS, Functional Genomics, Neurology, Pediatric surgery, and Amsterdam Neuroscience - Cellular & Molecular Mechanisms
- Subjects
0301 basic medicine ,In silico ,Protein subunit ,Mutation, Missense ,macromolecular substances ,QH426-470 ,030105 genetics & heredity ,Molecular Dynamics Simulation ,medicine.disease_cause ,eIF2B mutations ,03 medical and health sciences ,3D model structure ,Protein Domains ,Leukoencephalopathies ,Genetics ,medicine ,Missense mutation ,Humans ,Molecular Biology ,Gene ,Genetics (clinical) ,Mutation ,Leukoencephalopathies/genetics ,biology ,Leukodystrophy ,Original Articles ,genotype–phenotype correlation ,Eukaryotic Initiation Factor-2B/genetics ,medicine.disease ,Phenotype ,Eukaryotic Initiation Factor-2B ,030104 developmental biology ,vanishing white matter ,eIF2B ,biology.protein ,Original Article ,Missense - Abstract
Background Vanishing white matter (VWM) is a leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)‐subunit genes (EIF2B1–EIF2B5); 80% are missense mutations. Clinical severity is highly variable, with a strong, unexplained genotype–phenotype correlation. Materials and Methods With information from a recent natural history study, we severity‐graded 97 missense mutations. Using in silico modeling, we created a new human eIF2B model structure, onto which we mapped the missense mutations. Mutated residues were assessed for location in subunits, eIF2B complex, and functional domains, and for information on biochemical activity. Results Over 50% of mutations have (ultra‐)severe phenotypic effects. About 60% affect the ε‐subunit, containing the catalytic domain, mostly with (ultra‐)severe effects. About 55% affect subunit cores, with variable clinical severity. About 36% affect subunit interfaces, mostly with severe effects. Very few mutations occur on the external eIf2B surface, perhaps because they have minor functional effects and are tolerated. One external surface mutation affects eIF2B‐substrate interaction and is associated with ultra‐severe phenotype. Conclusion Mutations that lead to (ultra‐)severe disease mostly affect amino acids with pivotal roles in complex formation and function of eIF2B. Therapies for VWM are emerging and reliable mutation‐based phenotype prediction is required for propensity score matching for trials and in the future for individualized therapy decisions., Vanishing white matter (VWM) is a clinically highly variable leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)‐subunit genes, mostly missense mutations. To gain insight into the strong genotype‐phenotype correlation, we severity‐graded 97 missense mutations using information from a clinical natural history study, created a new human eIF2B model structure by in silico modeling, and assessed mutated residues for location in subunits, eIF2B complex, and functional domains, and for information on biochemical activity. We demonstrated that mutations associated with (ultra‐)severe disease mostly affect amino acids with pivotal roles in complex formation and eIF2B function, while mutations on the external eIf2B surface are mostly associated with mild phenotypes, probably because of minor functional effects. Therapies for VWM are emerging and reliable mutation‐based phenotype prediction is required for propensity score matching for trials and individualized therapy decisions.
- Published
- 2021
29. Understanding the Mechanistic Requirements for Efficient and Stereoselective Alkene Epoxidation by a Cytochrome P450 Enzyme
- Author
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Paul V. Bernhardt, Joshua S. Harbort, Alicia M. Kirk, Stephen Bell, M.N. Podgorski, Elizabeth H. Krenske, Luke R. Churchman, Rebecca R. Chao, Jeffrey Harmer, John B. Bruning, T. Coleman, and James J. De Voss
- Subjects
chemistry.chemical_classification ,010405 organic chemistry ,Chemistry ,Alkene ,Stereochemistry ,Reactive intermediate ,Epoxide ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Catalysis ,0104 chemical sciences ,Hydroxylation ,chemistry.chemical_compound ,Catalytic cycle ,Stereoselectivity ,Chemoselectivity - Abstract
The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.
- Published
- 2021
30. A cell permeable bimane-constrained PCNA-interacting peptide
- Author
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Wayne D. Tilley, Andrew D. Abell, Zoya Kikhtyak, Beth A. Vandborg, Theresa E. Hickey, John B. Bruning, Denis B. Scanlon, and Aimee J. Horsfall
- Subjects
chemistry.chemical_classification ,DNA clamp ,010405 organic chemistry ,Peptidomimetic ,Peptide ,010402 general chemistry ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,0104 chemical sciences ,Chemistry ,chemistry.chemical_compound ,chemistry ,Bimane ,Chemistry (miscellaneous) ,Biophysics ,Molecular Biology ,Linker ,Protein secondary structure ,Fluorescent tag ,Cysteine - Abstract
The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 310-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143–151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with KD values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 310-helical binding conformation. This suggests the 310-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications., A small, inherently fluorescent macrocyclic peptide constrained with a bimane-linker is cell permeable, and binds the human sliding clamp protein, PCNA, in a 310-helical conformation with nanomolar affinity.
- Published
- 2021
31. The catalytic activity and structure of the lipid metabolizing CYP124 cytochrome P450 enzyme from Mycobacterium marinum
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Amna Ghith, John B. Bruning, and Stephen G. Bell
- Subjects
Biophysics ,Molecular Biology ,Biochemistry - Published
- 2023
32. A comparison of the bacterial CYP51 cytochrome P450 enzymes from Mycobacterium marinum and Mycobacterium tuberculosis
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Hebatalla Mohamed, Stella A. Child, John B. Bruning, and Stephen G. Bell
- Subjects
Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Cell Biology ,Mycobacterium tuberculosis ,Ligands ,Biochemistry ,Lanosterol ,Sterol 14-Demethylase ,Endocrinology ,Bacterial Proteins ,Cytochrome P-450 Enzyme System ,Mycobacterium marinum ,Molecular Medicine ,Molecular Biology - Abstract
Members of the CYP51 family of cytochrome P450 enzymes are classified as sterol demethylases involved in the metabolic formation of cholesterol and related derivatives. The CYP51 enzyme from Mycobacterium marinum was studied and compared to its counterpart from Mycobacterium tuberculosis to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding of the purified CYP51 enzymes from M. marinum and M. tuberculosis were performed. The catalytic oxidation of lanosterol and related steroids was investigated. M. marinum CYP51 was structurally characterized by X-ray crystallography. The CYP51 enzyme of M. marinum is sequentially closely related to CYP51B1 from M. tuberculosis. However, differences in the heme spin state of each enzyme were observed upon the addition of steroids and other ligands. Both enzymes displayed different binding properties to those reported for the CYP51-Fdx fusion protein from the bacterium Methylococcus capsulatus. The enzymes were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol, but no products were detected for the related species dihydrolanosterol and eburicol. The crystal structure of CYP51 from M. marinum in the absence of added substrate but with a Bis-Tris molecule within the active site was resolved. The CYP51 enzyme of M. marinum displays differences in how steroids and other ligands bind compared to the M. tuberculosis enzyme. This was related to structural differences between the two enzymes. Overall, both of these CYP51 enzymes from mycobacterial species displayed significant differences to the CYP51 enzymes of eukaryotic species and the bacterial CYP51-Fdx enzyme of Me. capsulatus.
- Published
- 2022
33. d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations
- Author
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Jordan L. Pederick, Stephen Bell, John B. Bruning, and Andrew P. Thompson
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Models, Molecular ,0301 basic medicine ,Stereochemistry ,Biochemistry ,03 medical and health sciences ,Adenosine Triphosphate ,Enzyme kinetics ,Peptide Synthases ,Binding site ,Molecular Biology ,chemistry.chemical_classification ,DNA ligase ,030102 biochemistry & molecular biology ,biology ,Metals, Alkali ,Chemistry ,Thermus thermophilus ,Active site ,Cell Biology ,Cations, Monovalent ,biology.organism_classification ,D-alanine—D-alanine ligase ,Enzyme structure ,body regions ,030104 developmental biology ,Enzyme ,Biocatalysis ,Enzymology ,biology.protein - Abstract
The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K(+), but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K(+) and Rb(+) ions. A strict dependence on ionic radius of the MVC was observed, with Li(+) and Na(+) providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K(+), Rb(+), or Cs(+). Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.
- Published
- 2020
34. Structural insights into the role of the acid-alcohol pair of residues required for dioxygen activation in cytochrome P450 enzymes
- Author
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Stephen Bell, T. Coleman, Jeanette E. Stok, M.N. Podgorski, John B. Bruning, and James J. De Voss
- Subjects
Models, Molecular ,Stereochemistry ,chemistry.chemical_element ,Crystallography, X-Ray ,010402 general chemistry ,Benzoates ,01 natural sciences ,Biochemistry ,Oxygen ,Inorganic Chemistry ,chemistry.chemical_compound ,Residue (chemistry) ,Cytochrome P-450 Enzyme System ,Humans ,Threonine ,Heme ,chemistry.chemical_classification ,Molecular Structure ,biology ,010405 organic chemistry ,Chemistry ,Hydrogen bond ,Cytochrome P450 ,Monooxygenase ,0104 chemical sciences ,Enzyme ,Alcohols ,biology.protein - Abstract
The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4D251N and CYP199A4T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s. Graphic abstract
- Published
- 2020
35. Targeting PCNA with Peptide Mimetics for Therapeutic Purposes
- Author
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John B. Bruning, Andrew D. Abell, and Aimee J. Horsfall
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Models, Molecular ,Peptidomimetic ,Peptide ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Protein Domains ,Proliferating Cell Nuclear Antigen ,Humans ,Molecular Biology ,chemistry.chemical_classification ,DNA clamp ,biology ,010405 organic chemistry ,Conserved motif ,Organic Chemistry ,DNA replication ,0104 chemical sciences ,Proliferating cell nuclear antigen ,Cell biology ,Structural biology ,chemistry ,biology.protein ,Molecular Medicine ,Peptides ,Shut down ,Protein Binding - Abstract
Proliferating cell nuclear antigen (PCNA) is an excellent inhibition target to shut down highly proliferative cells and thereby develop a broad-spectrum cancer therapeutic. It interacts with a wide variety of proteins through a conserved motif referred to as the PCNA-interacting protein (PIP) box. There is large sequence diversity between high-affinity PCNA binding partners, but with conservation of the binding structure-a well-defined 310 -helix. Herein, all current PIP-box peptides crystallised with human PCNA are collated to reveal common trends between binding structure and affinity. Key intra- and intermolecular hydrogen-bonding networks that stabilise the 310 -helix of PIP-box partners are highlighted and related back to the canonical PIP-box motif. High correlation with the canonical PIP-box sequence does not directly afford high affinity. Instead, we summarise key interactions that stabilise the binding structure that leads to enhanced PCNA binding affinity. These interactions also implicate the "non-conserved" residues within the PIP-box that have previously been overlooked. Such insights will allow a more directed approach to develop therapeutic PCNA inhibitors.
- Published
- 2019
36. Inhibition of
- Author
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Nicholas C, Schumann, Kwang Jun, Lee, Andrew P, Thompson, Wanisa, Salaemae, Jordan L, Pederick, Thomas, Avery, Birgit I, Gaiser, James, Hodgkinson-Bean, Grant W, Booker, Steven W, Polyak, John B, Bruning, Kate L, Wegener, and Andrew D, Abell
- Subjects
Drug Development ,Molecular Structure ,Antitubercular Agents ,Carbon-Nitrogen Ligases ,Mycobacterium tuberculosis ,Enzyme Inhibitors ,Crystallography, X-Ray ,Protein Binding - Published
- 2021
37. Approaches to Introduce Helical Structure in Cysteine-Containing Peptides with a Bimane Group
- Author
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Aimee J. Horsfall, John B. Bruning, Denis B. Scanlon, Andrew D. Abell, and Daniel P. McDougal
- Subjects
Protein Conformation, alpha-Helical ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Peptidomimetic ,Peptide ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Residue (chemistry) ,chemistry.chemical_compound ,Bimane ,Amino Acid Sequence ,Cysteine ,Molecular Biology ,Alanine ,chemistry.chemical_classification ,Peptide modification ,010405 organic chemistry ,Circular Dichroism ,Organic Chemistry ,Estrogen Receptor alpha ,Bridged Bicyclo Compounds, Heterocyclic ,0104 chemical sciences ,Peptide Conformation ,Spectrometry, Fluorescence ,chemistry ,Molecular Medicine ,Peptides ,Linker ,Protein Binding - Abstract
An i-i+4 or i-i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i-i+4 constrained peptide adopts a 310 -helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.
- Published
- 2021
38. An aldo-keto reductase with 2-keto-l-gulonate reductase activity functions in l-tartaric acid biosynthesis from vitamin C in Vitis vinifera
- Author
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Robert D. Hancock, John B. Bruning, Yong Jia, Crista A. Burbidge, Colin L. D. Jenkins, Kathy Soole, Crystal Sweetman, Christopher M. Ford, and Emi Schutz
- Subjects
0301 basic medicine ,Antioxidant ,protein crystallization ,substrate specificity ,medicine.medical_treatment ,2-keto-L-gulonic acid ,Glyoxylate cycle ,Aldo-Keto Reductases ,Plant Biology ,Ascorbic Acid ,Reductase ,Biochemistry ,Wine grape ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,aldo-keto reductase ,Biosynthesis ,enzyme kinetics ,Catalytic Domain ,Pyruvic Acid ,medicine ,structural biology ,enzyme mechanism ,Vitis ,Molecular Biology ,Tartrates ,X-ray crystallography ,Plant Proteins ,Aldo-keto reductase ,tartaric acid synthesis ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Glyoxylates ,Sugar Acids ,Cell Biology ,Ascorbic acid ,grapevine ,030104 developmental biology ,plant biochemistry ,Vitis vinifera ,docking ,biology.protein ,D-isomer-specific 2-hydroxyacid dehydrogenase - Abstract
Tartaric acid has high economic value as an antioxidant and flavorant in food and wine industries. l-Tartaric acid biosynthesis in wine grape (Vitis vinifera) uses ascorbic acid (vitamin C) as precursor, representing an unusual metabolic fate for ascorbic acid degradation. Reduction of the ascorbate breakdown product 2-keto-l-gulonic acid to l-idonic acid constitutes a critical step in this l-tartaric acid biosynthetic pathway. However, the underlying enzymatic mechanisms remain obscure. Here, we identified a V. vinifera aldo-keto reductase, Vv2KGR, with 2-keto-l-gulonic acid reductase activity. Vv2KGR belongs to the d-isomer–specific 2-hydroxyacid dehydrogenase superfamily and displayed the highest similarity to the hydroxyl pyruvate reductase isoform 2 in Arabidopsis thaliana. Enzymatic analyses revealed that Vv2KGR efficiently reduces 2-keto-l-gulonic acid to l-idonic acid and uses NADPH as preferred coenzyme. Moreover, Vv2KGR exhibited broad substrate specificity toward glyoxylate, pyruvate, and hydroxypyruvate, having the highest catalytic efficiency for glyoxylate. We further determined the X-ray crystal structure of Vv2KGR at 1.58 Å resolution. Comparison of the Vv2KGR structure with those of d-isomer–specific 2-hydroxyacid dehydrogenases from animals and microorganisms revealed several unique structural features of this plant hydroxyl pyruvate reductase. Substrate structural analysis indicated that Vv2KGR uses two modes (A and B) to bind different substrates. 2-Keto-l-gulonic acid displayed the lowest predicted free-energy binding to Vv2KGR among all docked substrates. Hence, we propose that Vv2KGR functions in l-tartaric acid biosynthesis. To the best of our knowledge, this is the first report of a d-isomer–specific 2-hydroxyacid dehydrogenase that reduces 2-keto-l-gulonic acid to l-idonic acid in plants.
- Published
- 2019
39. Sulfonamide-Based Inhibitors of Biotin Protein Ligase as New Antibiotic Leads
- Author
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Kwangjun Lee, Andrew D. Abell, Steven W. Polyak, Beatriz Blanco-Rodriguez, John B. Bruning, Danielle Cini, Grant W. Booker, Robert W. Milne, Benjamin Noll, Andrew J. Hayes, Andrew C Marshall, Ashleigh S. Paparella, Jiage Feng, Matthew C.J. Wilce, Jingxian Yu, William Tieu, Lee, Kwang Jun, Tieu, William, Blanco-Rodriguez, Beatriz, Paparella, Ashleigh S, Yu, Jingxian, Hayes, Andrew, Feng, Jiage, Marshall, Andrew C, Noll, Benjamin, Milne, Robert, Cini, Danielle, Wilce, Matthew CJ, Booker, Grant W, Bruning, John B, Polyak, Steven W, and Abell, Andrew D
- Subjects
pharmacokinetic profile ,0301 basic medicine ,Staphylococcus aureus ,medicine.drug_class ,Antibiotics ,Microbial Sensitivity Tests ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,metabolic stability ,Microbiology ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Drug Stability ,Biotin ,medicine ,Animals ,Carbon-Nitrogen Ligases ,Enzyme Inhibitors ,chemistry.chemical_classification ,Sulfonamides ,DNA ligase ,010405 organic chemistry ,molecular dynamics simulations ,General Medicine ,Phosphate ,Anti-Bacterial Agents ,Rats ,0104 chemical sciences ,Sulfonamide ,030104 developmental biology ,chemistry ,Drug Design ,Molecular Medicine - Abstract
Here, we report the design, synthesis, and evaluation of a series of inhibitors of Staphylococcus aureus BPL (SaBPL), where the central acyl phosphate of the natural intermediate biotinyl-5′-AMP (1) is replaced by a sulfonamide isostere. Acylsulfamide (6) and amino sulfonylurea (7) showed potent in vitro inhibitory activity (Ki = 0.007 ± 0.003 and 0.065 ± 0.03 μM, respectively) and antibacterial activity against S. aureus ATCC49775 with minimum inhibitory concentrations of 0.25 and 4 μg/mL, respectively. Additionally, the bimolecular interactions between the BPL and inhibitors 6 and 7 were defined by X-ray crystallography and molecular dynamics simulations. The high acidity of the sulfonamide linkers of 6 and 7 likely contributes to the enhanced in vitro inhibitory activities by promoting interaction with SaBPL Lys187. Analogues with alkylsulfamide (8), β-ketosulfonamide (9), and β-hydroxysulfonamide (10) isosteres were devoid of significant activity. Binding free energy estimation using computational methods suggests deprotonated 6 and 7 to be the best binders, which is consistent with enzyme assay results. Compound 6 was unstable in whole blood, leading to poor pharmacokinetics. Importantly, 7 has a vastly improved pharmacokinetic profile compared to that of 6 presumably due to the enhanced metabolic stability of the sulfonamide linker moiety. Refereed/Peer-reviewed
- Published
- 2019
40. Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68
- Author
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John B. Bruning, Jia Quyen Truong, Santosh Panjikar, Keith E. Shearwin, and Linda M. Shearwin-Whyatt
- Subjects
Models, Molecular ,0303 health sciences ,Materials science ,biology ,030302 biochemistry & molecular biology ,Lysin ,Crystallography, X-Ray ,Ring (chemistry) ,biology.organism_classification ,Phaser ,Bacteriophage ,Viral Proteins ,03 medical and health sciences ,Crystallography ,Matrix (mathematics) ,Structural Biology ,Triiodobenzoic Acids ,Endopeptidases ,Hydrolase ,Domain (ring theory) ,Molecule ,Muramidase ,Staphylococcus Phages ,Crystallization ,030304 developmental biology - Abstract
Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein fromStaphylococcusphage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.
- Published
- 2019
41. Shooting three inflammatory targets with a single bullet: Novel multi-targeting anti-inflammatory glitazones
- Author
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Benjamin S. K. Chua, Perihan A. Elzahhar, Tamer M. Ibrahim, Rasha A. Nassra, Rana Alaaeddine, Ahmed F. El-Yazbi, Hala F. Labib, Nadja Wallner, Ahmed S.F. Belal, John B. Bruning, Rebecca L. Frkic, Azza Ismail, Tilo Knape, and Andreas von Knethen
- Subjects
medicine.drug_class ,medicine.medical_treatment ,In silico ,Anti-Inflammatory Agents ,Inflammation ,Pharmacology ,Ligands ,Anti-inflammatory ,Drug Discovery ,medicine ,Animals ,Arachidonate 15-Lipoxygenase ,Humans ,Ligand efficiency ,Cyclooxygenase 2 Inhibitors ,Chemistry ,Monocyte ,Organic Chemistry ,General Medicine ,Molecular Docking Simulation ,PPAR gamma ,medicine.anatomical_structure ,Cytokine ,Drug development ,Docking (molecular) ,Drug Design ,Thiazolidinediones ,medicine.symptom ,Protein Binding - Abstract
In search for effective multi-targeting drug ligands (MTDLs) to address low-grade inflammatory changes of metabolic disorders, we rationally designed some novel glitazones-like compounds. This was achieved by incorporating prominent pharmacophoric motifs from previously reported COX-2, 15-LOX and PPARγ ligands. Challenging our design with pre-synthetic docking experiments on PPARγ showed encouraging results. In vitro tests have identified 4 compounds as simultaneous partial PPARγ agonist, potent COX-2 antagonist (nanomolar IC50 values) and moderate 15-LOX inhibitor (micromolar IC50 values). We envisioned such outcome as a prototypical balanced modulation of the 3 inflammatory targets. In vitro glucose uptake assay defined six compounds as insulin-sensitive and the other two as insulin-independent glucose uptake enhancers. Also, they were able to induce PPARγ nuclear translocation in immunohistochemical analysis. Their anti-inflammatory potential has been translated to effective inhibition of monocyte to macrophage differentiation, suppression of LPS-induced inflammatory cytokine production in macrophages, as well as significant in vivo anti-inflammatory activity. Ligand co-crystallized PPARγ X-ray of one of MTDLs has identified new clues that could serve as structural basis for its partial agonism. Docking of the most active compounds into COX-2 and 15-LOX active sites, pinpointed favorable binding patterns, similar to those of the co-crystallized ligands. Finally, in silico assessment of pharmacokinetics, physicochemical properties, drug-likeness and ligand efficiency indices was performed. Hence, we anticipate that the prominent biological profile of such series will rationalize relevant anti-inflammatory drug development endeavors.
- Published
- 2019
42. The role of N-terminal heterocycles in hydrogen bonding to α-chymotrypsin
- Author
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Andrew D. Abell, Andrew C Marshall, Nicholas C Schumann, and John B. Bruning
- Subjects
Serine Proteinase Inhibitors ,Stereochemistry ,Clinical Biochemistry ,Pharmaceutical Science ,01 natural sciences ,Biochemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Amide ,Furan ,Drug Discovery ,Pyridine ,Thiophene ,Chymotrypsin ,Pyrroles ,Molecular Biology ,Pyrrole ,Aldehydes ,Dipeptide ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,010405 organic chemistry ,Chemistry ,Hydrogen bond ,Organic Chemistry ,Hydrogen Bonding ,Dipeptides ,0104 chemical sciences ,Molecular Docking Simulation ,010404 medicinal & biomolecular chemistry ,biology.protein ,Molecular Medicine - Abstract
A series of dipeptide aldehydes containing different N-terminal heterocycles was prepared and assayed in vitro against α-chymotrypsin to ascertain the importance of the heterocycle in maintaining a β-strand geometry while also providing a hydrogen bond donor equivalent to the backbone amide nitrogen of the surrogate amino acid. The dipeptide containing a pyrrole constraint (10) was the most potent inhibitor, with >30-fold improved activity over dipeptides which lacked a nitrogen hydrogen bond donor (namely thiophene 11, furan 12 and pyridine 13). Molecular docking studies of 10 bound to α-chymotrypsin demonstrates a hydrogen bond between the pyrrole nitrogen donor and the backbone carbonyl of Gly216 located in the S3 pocket which is proposed to be critical for overall binding.
- Published
- 2019
43. Precipitant–ligand exchange technique reveals the ADP binding mode inMycobacterium tuberculosisdethiobiotin synthetase
- Author
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Steven W. Polyak, Andrew P. Thompson, Kate L. Wegener, Grant W. Booker, John B. Bruning, Thompson, Andrew P, Wegener, Kate L, Booker, Grant W, Polyak, Steven W, and Bruning, John B
- Subjects
0301 basic medicine ,Biochemistry & Molecular Biology ,Cytidine triphosphate ,Protein Conformation ,Cytidine Triphosphate ,Biophysics ,Plasma protein binding ,ligand ,Crystallography, X-Ray ,Ligands ,Binding, Competitive ,Biochemical Research Methods ,Mycobacterium tuberculosis ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,Transferase ,Carbon-Nitrogen Ligases ,Binding site ,Crystallography ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Chemistry ,exchange ,biology.organism_classification ,Combinatorial chemistry ,Adenosine Diphosphate ,Adenosine diphosphate ,030104 developmental biology ,dethiobiotin synthetase ,ADP binding ,Protein Binding - Abstract
Dethiobiotin synthetase fromMycobacterium tuberculosis(MtDTBS) is a promising antituberculosis drug target. Small-molecule inhibitors that targetMtDTBS provide a route towards new therapeutics for the treatment of antibiotic-resistant tuberculosis. Adenosine diphosphate (ADP) is an inhibitor ofMtDTBS; however, structural studies into its mechanism of inhibition have been unsuccessful owing to competitive binding to the enzyme by crystallographic precipitants such as citrate and sulfate. Here, a crystallographic technique termed precipitant–ligand exchange has been developed to exchange protein-bound precipitants with ligands of interest. Proof of concept for the exchange method was demonstrated using cytidine triphosphate (CTP), which adopted the same binding mechanism as that obtained with traditional crystal-soaking techniques. Precipitant–ligand exchange also yielded the previously intractable structure ofMtDTBS in complex with ADP solved to 2.4 Å resolution. This result demonstrates the utility of precipitant–ligand exchange, which may be widely applicable to protein crystallography.
- Published
- 2018
44. Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid
- Author
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Stephanie Nguyen, John B. Bruning, Keith E. Shearwin, and Jia Q. Truong
- Subjects
Models, Molecular ,Materials science ,Protein Conformation ,030303 biophysics ,Biophysics ,Phthalic Acids ,Crystal structure ,Phase problem ,Crystallography, X-Ray ,Biochemistry ,Method Communications ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,law ,Atom ,Genetics ,Animals ,Molecular replacement ,Crystallization ,Derivatization ,030304 developmental biology ,0303 health sciences ,Condensed Matter Physics ,Phaser ,Hydrocarbons, Brominated ,chemistry ,Chemical physics ,Muramidase ,Chickens ,Macromolecule - Abstract
The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.
- Published
- 2021
45. Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening
- Author
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John B. Bruning, Jia Quyen Truong, Stephanie Nguyen, and Keith E. Shearwin
- Subjects
Data Analysis ,Models, Molecular ,Diffraction ,Materials science ,General Chemical Engineering ,Crystal structure ,Phase problem ,Lithium ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Diffusion ,Matrix (mathematics) ,Imaging, Three-Dimensional ,Protein structure ,Triiodobenzoic Acids ,Animals ,Molecule ,Quantitative Biology::Biomolecules ,General Immunology and Microbiology ,General Neuroscience ,Proteins ,Chemical physics ,X-ray crystallography ,Muramidase ,Protein crystallization ,Chickens - Abstract
Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.
- Published
- 2021
46. Targeting Unconventional Pathways in Pursuit of Novel Antifungals
- Author
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Stephanie Nguyen, John B. Bruning, and Jia Q. Truong
- Subjects
0301 basic medicine ,030106 microbiology ,Antifungal drug ,Cryptococcus ,Computational biology ,Review ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,drug discovery ,03 medical and health sciences ,Candida albicans ,drug targets ,Molecular Biosciences ,lcsh:QH301-705.5 ,Molecular Biology ,Cryptococcus neoformans ,chemistry.chemical_classification ,Aspergillus ,biology ,Drug discovery ,Aspergillus fumigatus ,biology.organism_classification ,Metabolic pathway ,030104 developmental biology ,Enzyme ,lcsh:Biology (General) ,chemistry ,antifungal - Abstract
The impact of invasive fungal infections on human health is a serious, but largely overlooked, public health issue. Commonly affecting the immunocompromised community, fungal infections are predominantly caused by species of Candida, Cryptococcus, and Aspergillus. Treatments are reliant on the aggressive use of pre-existing antifungal drug classes that target the fungal cell wall and membrane. Despite their frequent use, these drugs are subject to unfavorable drug-drug interactions, can cause undesirable side-effects and have compromised efficacy due to the emergence of antifungal resistance. Hence, there is a clear need to develop novel classes of antifungal drugs. A promising approach involves exploiting the metabolic needs of fungi by targeted interruption of essential metabolic pathways. This review highlights potential antifungal targets including enolase, a component of the enolase-plasminogen complex, and enzymes from the mannitol biosynthesis and purine nucleotide biosynthesis pathways. There has been increased interest in the enzymes that comprise these particular pathways and further investigation into their merits as antifungal targets and roles in fungal survival and virulence are warranted. Disruption of these vital processes by targeting unconventional pathways with small molecules or antibodies may serve as a promising approach to discovering novel classes of antifungals.
- Published
- 2021
47. PPARα and δ Ligand Design: Honing the Traditional Empirical Method with a More Holistic Overview
- Author
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John B. Bruning and Benjamin S. K. Chua
- Subjects
chemistry.chemical_classification ,Clinical therapy ,chemistry ,Ligand ,Peroxisome proliferator-activated receptor ,Computational biology ,Metabolic disease - Abstract
Peroxisome proliferator-activated receptor (PPAR) ligands have been used in clinical therapy to treat metabolic disease since the 1960s. However, these ligands have side effects that restrict their use, thought to be caused, in part, by their broad specificity. Efforts have been made to synthesize new ligands; however, most have failed to pass clinical trials. Here we examine the available crystal structures of PPAR in complex with ligands to identify common ligand design factors for selectivity towards a PPAR subtype. Methods to improve drug-lead identification and optimization and other factors that may contribute to design of a successful PPAR ligand are discussed.
- Published
- 2021
48. Nucleoside selectivity of Aspergillus fumigatus nucleoside-diphosphate kinase
- Author
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Blagojce Jovcevski, Tara L. Pukala, John B. Bruning, and Stephanie Nguyen
- Subjects
0301 basic medicine ,Specificity constant ,Protein Conformation ,Protein Data Bank (RCSB PDB) ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Escherichia coli ,Aspergillosis ,Humans ,Phosphorylation ,Molecular Biology ,chemistry.chemical_classification ,Aspergillus fumigatus ,Cytidine ,Nucleosides ,Cell Biology ,Nucleoside-diphosphate kinase ,Kinetics ,030104 developmental biology ,Enzyme ,chemistry ,Structural biology ,030220 oncology & carcinogenesis ,Nucleoside-Diphosphate Kinase ,Nucleoside ,DNA - Abstract
Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s-1 ), specificity constant (626 ± 110.0 mm-1 ·s-1 ) and binding free energy change (-37.0 ± 3.5 kcal·mol-1 ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s-1 ) and lowest specificity constant (40.2 ± 4.4 mm-1 ·s-1 ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV).
- Published
- 2020
49. TSC-Insensitive Rheb Mutations Induce Oncogenic Transformation Through a Combination of Hyperactive mTORC1 Signalling and Metabolic Reprogramming
- Author
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Stuart P. De Poi, John B. Bruning, Christopher G. Proud, Wenru Pan, Sean J. Humphrey, Leanne K. Hein, Jianling Xie, and Timothy J. Sargeant
- Subjects
Cell growth ,Mutant ,Autophagy ,biology.protein ,Regulator ,mTORC1 ,Translation factor ,Biology ,EEF2 ,Cell biology ,RHEB - Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1, that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, Tuberous Sclerosis Complex.We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving metabolic reprogramming and an increased rate of glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through a differential interaction with AMPK.Our findings suggest that unique ‘bespoke’ combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.
- Published
- 2020
50. TSC-insensitive Rheb mutations induce oncogenic transformation through a combination of constitutively active mTORC1 signalling and proteome remodelling
- Author
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Jianling, Xie, Stuart P, De Poi, Sean J, Humphrey, Leanne K, Hein, John B, Bruning, Wenru, Pan, Luke A, Selth, Timothy J, Sargeant, and Christopher G, Proud
- Subjects
Models, Molecular ,Proteomics ,Proteome ,Mechanistic Target of Rapamycin Complex 1 ,Tuberous Sclerosis Complex 1 Protein ,Mice ,HEK293 Cells ,Neoplasms ,Tuberous Sclerosis Complex 2 Protein ,NIH 3T3 Cells ,Animals ,Humans ,Point Mutation ,Ras Homolog Enriched in Brain Protein ,HeLa Cells ,Signal Transduction - Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5' AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.
- Published
- 2020
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