8 results on '"David K. Miyamoto"'
Search Results
2. Discovery of a Celecoxib Binding Site on Prostaglandin E Synthase (PTGES) with a Cleavable Chelation-Assisted Biotin Probe
- Author
-
Christina M. Woo, Hope A. Flaxman, David K. Miyamoto, Jinxu Gao, and Hung-Yi Wu
- Subjects
0301 basic medicine ,Biotin ,Prostaglandin ,Photoaffinity Labels ,Prostaglandin E synthase ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Binding site ,Chelating Agents ,Prostaglandin-E Synthases ,Binding Sites ,Cyclooxygenase 2 Inhibitors ,biology ,Photoaffinity labeling ,010405 organic chemistry ,General Medicine ,Small molecule ,0104 chemical sciences ,Cell biology ,030104 developmental biology ,chemistry ,Membrane protein ,Celecoxib ,Molecular Probes ,Prostaglandins ,biology.protein ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,Signal transduction ,Signal Transduction - Abstract
The coxibs are a subset of nonsteroidal anti-inflammatory drugs (NSAIDs) that primarily target cyclooxygenase-2 (COX-2) to inhibit prostaglandin signaling and reduce inflammation. However, mechanisms to inhibit other members of the prostaglandin signaling pathway may improve selectivity and reduce off-target toxicity. Here, we report a novel binding site for celecoxib on prostaglandin E synthase (PTGES), which is an enzyme downstream of COX-2 in the prostaglandin signaling pathway, using a cleavable chelation-assisted biotin probe 6. Evaluation of the multifunctional probe 6 revealed significantly improved tagging efficiencies attributable to the embedded picolyl functional group. Application of the probe 6 within the small molecule interactome mapping by photoaffinity labeling (SIM-PAL) platform using photo-celecoxib as a reporter for celecoxib identified PTGES and other membrane proteins in the top eight enriched proteins from A549 cells. Four binding sites to photo-celecoxib were mapped by the probe 6, including a binding site with PTGES. The binding interaction with PTGES was validated by competitive displacement with celecoxib and licofelone, which is a known PTGES inhibitor, and was used to generate a structural model of the interaction. The identification of photo-celecoxib interactions with membrane proteins, including the direct binding site on the membrane protein PTGES, will inform further functional followup and the design of new selective inhibitors of the prostaglandin signaling pathway.
- Published
- 2019
3. Small Molecule Interactome Mapping by Photo‐Affinity Labeling (SIM‐PAL) to Identify Binding Sites of Small Molecules on a Proteome‐Wide Scale
- Author
-
Christina M. Woo, Hope A. Flaxman, and David K. Miyamoto
- Subjects
Proteomics ,0301 basic medicine ,Azides ,Proteome ,Ultraviolet Rays ,Quantitative proteomics ,010402 general chemistry ,01 natural sciences ,Interactome ,Catalysis ,Mass Spectrometry ,Article ,Cell Line ,Small Molecule Libraries ,03 medical and health sciences ,chemistry.chemical_compound ,Biotin ,Humans ,Non-covalent interactions ,Biotinylation ,Binding site ,chemistry.chemical_classification ,Binding Sites ,Affinity labeling ,Cycloaddition Reaction ,Staining and Labeling ,Chemistry ,Proteins ,General Medicine ,Small molecule ,0104 chemical sciences ,030104 developmental biology ,Biochemistry ,Alkynes ,Protein Binding - Abstract
Identification and characterization of small molecule-protein interactions is critical to understanding the mechanism of action of bioactive small molecules. Photo-affinity labeling (PAL) enables the capture of noncovalent interactions for enrichment and unbiased analysis by mass spectrometry (MS). Quantitative proteomics of the enriched proteome reveals potential interactions, and MS characterization of binding sites provides validation and structural insight into the interactions. Here, we describe the identification of the protein targets and binding sites of a small molecule using small molecule interactome mapping by PAL (SIM-PAL). Cells are exposed to a diazirine-alkyne-functionalized small molecule, and binding interactions are covalently captured upon UV irradiation. An isotopically coded, acid-cleavable biotin azide handle is attached to the conjugated proteins using copper-catalyzed azide-alkyne cycloaddition. Biotin-labeled proteins are enriched for on-bead digestion and quantitative proteomics. Acid cleavage of the handle releases the bead-bound conjugated peptides for MS analysis and isotope-directed assignment of the binding site. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a small molecule-conjugated protein sample following treatment of live cells Alternate Protocol: Generation of a small molecule-conjugated protein sample following treatment of cell lysate Basic Protocol 2: Copper-catalyzed azide-alkyne cycloaddition functionalization and enrichment of labeled peptides Support Protocol 1: Synthesis of acid-cleavable, isotopically coded biotin picolyl azide handle Support Protocol 2: Monitoring enrichment by immunoblotting Basic Protocol 3: Mass spectrometry analysis to identify interacting proteins and conjugation sites.
- Published
- 2019
4. Chemoproteomic Screening of Covalent Ligands Reveals UBA5 As a Novel Pancreatic Cancer Target
- Author
-
Carlo M. Contreras, Tucker R. Huffman, Ashley N. Ives, David Akopian, David K. Miyamoto, Allison M. Roberts, Daniel K. Nomura, Christine F. Skibola, Leslie A. Bateman, Martin J. Heslin, and Michael Rape
- Subjects
Proteomics ,0301 basic medicine ,Ubiquitin-activating enzyme ,Druggability ,Antineoplastic Agents ,Ubiquitin-Activating Enzymes ,Biology ,Ligands ,Polymerase Chain Reaction ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,Pancreatic cancer ,medicine ,Humans ,chemistry.chemical_classification ,010405 organic chemistry ,Ligand ,General Medicine ,medicine.disease ,Small molecule ,0104 chemical sciences ,Pancreatic Neoplasms ,030104 developmental biology ,Enzyme ,Mechanism of action ,chemistry ,Gene Knockdown Techniques ,Molecular Medicine ,medicine.symptom - Abstract
Chemical genetic screening of small-molecule libraries has been a promising strategy for discovering unique and novel therapeutic compounds. However, identifying the targets of lead molecules that arise from these screens has remained a major bottleneck in understanding the mechanism of action of these compounds. Here, we have coupled the screening of a cysteine-reactive fragment-based covalent ligand library with an isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) chemoproteomic platform to rapidly couple the discovery of lead small molecules that impair pancreatic cancer pathogenicity with the identification of druggable hotspots for potential cancer therapy. Through this coupled approach, we have discovered a covalent ligand DKM 2-93 that impairs pancreatic cancer cell survival and in vivo tumor growth through covalently modifying the catalytic cysteine of the ubiquitin-like modifier activating enzyme 5 (UBA5), thereby inhibiting its activity as a protein that activates the ubiquitin-like protein UFM1 to UFMylate proteins. We show that UBA5 is a novel pancreatic cancer therapeutic target and show DKM 2-93 as a relatively selective lead inhibitor of UBA5. Our results underscore the utility of coupling the screening of covalent ligand libraries with isoTOP-ABPP platforms for mining the proteome for druggable hotspots for cancer therapy.
- Published
- 2017
5. Discovery of a celecoxib binding site on PTGES with a cleavable chelation-assisted biotin probe
- Author
-
Christina M. Woo, Hope A. Flaxman, Jinxu Gao, Hung-Yi Wu, and David K. Miyamoto
- Subjects
genetic structures ,biology ,Chemistry ,Prostaglandin ,Prostaglandin E synthase ,Small molecule ,Cell biology ,chemistry.chemical_compound ,Membrane protein ,Biotin ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Binding site ,Signal transduction ,Licofelone - Abstract
The coxibs are a subset of non-steroidal anti-inflammatory drugs (NSAIDs) that primarily target cyclooxygenase-2 (COX-2) to inhibit prostaglandin signaling and reduce inflammation. However, mechanisms to inhibit other members of the prostaglandin signaling pathway may improve selectivity and reduce off-target toxicity. Here, we report a novel binding site for celecoxib on prostaglandin E synthase (PTGES), an enzyme downstream of COX-2 in the prostaglandin signaling pathway, using a cleavable chelation-assisted biotin probe 6. Evaluation of the multi-functional probe 6 revealed significantly improved tagging efficiencies attributable to the embedded picolyl functional group. Application of the probe 6 within the small molecule interactome mapping by photo-affinity labeling (SIM-PAL) platform using photo-celecoxib as a reporter for celecoxib identified PTGES and other membrane proteins in the top eight enriched proteins from A549 cells. Carbonic anhydrase 12, a known protein target of celecoxib, was also enriched. Four binding sites to photo-celecoxib were additionally mapped by the probe 6, including a binding site with PTGES. The binding interaction with PTGES was validated by competitive displacement with celecoxib and known PTGES inhibitor licofelone. The binding site of photo-celecoxib on PTGES enabled the development of a structural model of the interaction and will inform the design of new selective inhibitors of the prostaglandin signaling pathway.
- Published
- 2019
6. Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity
- Author
-
Wan-min Ku, Tucker R. Huffman, Allison M. Roberts, Truc B. Nguyen, Yana Petri, James A. Olzmann, Christine F. Skibola, Daniel K. Nomura, Carlo M. Contreras, Leslie A. Bateman, Martin J. Heslin, and David K. Miyamoto
- Subjects
0301 basic medicine ,Proteomics ,Nuclear Envelope ,Nogo Proteins ,Druggability ,Antineoplastic Agents ,Computational biology ,Endoplasmic Reticulum ,Ligands ,01 natural sciences ,Catalysis ,Article ,03 medical and health sciences ,Materials Chemistry ,2.1 Biological and endogenous factors ,Humans ,Chemoproteomics ,Cysteine ,Aetiology ,Cancer ,Acrylamide ,010405 organic chemistry ,Chemistry ,Endoplasmic reticulum ,Prevention ,Organic Chemistry ,Metals and Alloys ,General Chemistry ,Molecular biology ,Endoplasmic reticulum tubular network formation ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Colo-Rectal Cancer ,030104 developmental biology ,5.1 Pharmaceuticals ,Proteome ,Chemical Sciences ,Ceramics and Composites ,Development of treatments and therapeutic interventions ,Digestive Diseases ,Colorectal Neoplasms ,Chemical genetics ,Reticulon 4 ,Biotechnology - Abstract
Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.
- Published
- 2017
7. Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products
- Author
-
Jordan I. Kleinman, Jessica N. Spradlin, Elizabeth A. Grossman, Daniel K. Nomura, Leslie A. Bateman, Carl C. Ward, Tucker R. Huffman, and David K. Miyamoto
- Subjects
0301 basic medicine ,Proteome ,Clinical Biochemistry ,Druggability ,Ligands ,Biochemistry ,chemistry.chemical_compound ,withaferin ,Drug Discovery ,Protein Phosphatase 2 ,Cancer ,Tumor ,Activity-based proteomics ,PP2A ,PPP2R1A ,5.1 Pharmaceuticals ,MCF-7 Cells ,Molecular Medicine ,Female ,Development of treatments and therapeutic interventions ,Signal Transduction ,Chemical biology ,chemical biology ,Antineoplastic Agents ,Breast Neoplasms ,Computational biology ,Biology ,Cell Line ,03 medical and health sciences ,Cell Line, Tumor ,Breast Cancer ,medicine ,Humans ,Chemoproteomics ,Amino Acid Sequence ,Cysteine ,Molecular Biology ,Withanolides ,activity-based protein profiling ,Cell Proliferation ,Pharmacology ,Biological Products ,Natural product ,covalent ligand discovery ,protein phosphatase 2A ,Protein phosphatase 2 ,medicine.disease ,chemoproteomics ,030104 developmental biology ,chemistry ,Withaferin A ,Proto-Oncogene Proteins c-akt - Abstract
Summary Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy.
- Published
- 2017
8. GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity
- Author
-
Lisa A. Crawford, Lucky Ding, Tucker R. Huffman, Andrei Goga, Elizabeth A. Grossman, Eranthie Weerapana, Roman Camarda, Daniel K. Nomura, Sharon M. Louie, and David K. Miyamoto
- Subjects
0301 basic medicine ,Clinical Biochemistry ,Antineoplastic Agents ,Triple Negative Breast Neoplasms ,Biology ,medicine.disease_cause ,Biochemistry ,Article ,Dose-Response Relationship ,03 medical and health sciences ,GSTP1 ,Mice ,Experimental ,Structure-Activity Relationship ,Breast cancer ,Leucine ,Neoplasms ,Drug Discovery ,Breast Cancer ,medicine ,Tumor Cells, Cultured ,Animals ,Humans ,2.1 Biological and endogenous factors ,Enzyme Inhibitors ,Aetiology ,Molecular Biology ,Triple-negative breast cancer ,Cancer ,Nutrition ,Pharmacology ,Cultured ,Dose-Response Relationship, Drug ,Molecular Structure ,Triazines ,Lipid metabolism ,Neoplasms, Experimental ,medicine.disease ,Tumor Cells ,030104 developmental biology ,Cell metabolism ,Glutathione S-Transferase pi ,Cancer research ,Molecular Medicine ,Drug ,Carcinogenesis - Abstract
Breast cancers possess fundamentally altered metabolism that fuels their pathogenicity. While many metabolic drivers of breast cancers have been identified, the metabolic pathways that mediate breast cancer malignancy and poor prognosis are less well understood. Here, we used a reactivity-based chemoproteomic platform to profile metabolic enzymes that are enriched in breast cancer cell types linked to poor prognosis, including triple-negative breast cancer (TNBC) cells and breast cancer cells that have undergone an epithelial-mesenchymal transition-like state of heightened malignancy. We identified glutathione S-transferase Pi 1 (GSTP1) as a novel TNBC target that controls cancer pathogenicity by regulating glycolytic and lipid metabolism, energetics, and oncogenic signaling pathways through a protein interaction that activates glyceraldehyde-3-phosphate dehydrogenase activity. We show that genetic or pharmacological inactivation of GSTP1 impairs cell survival and tumorigenesis inTNBC cells. We put forth GSTP1 inhibitors as a noveltherapeutic strategy for combatting TNBCs through impairing key cancer metabolism and signaling pathways.
- Published
- 2016
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.