Villamor J.G.,Max Planck Institute for Plant Breeding Research |
Kaschani F.,Max Planck Institute for Plant Breeding Research |
Kaschani F.,University of Duisburg - Essen |
Colby T.,Max Planck Institute for Plant Breeding Research |
And 5 more authors.
Molecular and Cellular Proteomics | Year: 2013
Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
Dix M.M.,Scripps Research Institute |
Simon G.M.,Scripps Research Institute |
Wang C.,Scripps Research Institute |
Okerberg E.,ActivX Biosciences |
And 2 more authors.
Cell | Year: 2012
Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation. © 2012 Elsevier Inc.
Zhang T.,Dana-Farber Cancer Institute |
Zhang T.,Harvard University |
Inesta-Vaquera F.,University of Dundee |
Niepel M.,Harvard University |
And 20 more authors.
Chemistry and Biology | Year: 2012
The mitogen-activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here, we report the discovery of irreversible inhibitors of JNK1/2/3. We describe two JNK3 cocrystal structures at 2.60 and 2.97 resolution that show the compounds form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK, in cells exposed to submicromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochemical, cellular, and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggests that the compound will be broadly useful as a pharmacological probe of JNK-dependent signal transduction. Potential lead compounds have also been identified for kinases, including IRAK1, PIK3C3, PIP4K2C, and PIP5K3. © 2012 Elsevier Ltd All rights reserved.
Deng X.,Dana-Farber Cancer Institute |
Deng X.,Harvard University |
Dzamko N.,University of Dundee |
Prescott A.,University of Dundee |
And 10 more authors.
Nature Chemical Biology | Year: 2011
Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. We employed a new, parallel, compound-centric approach to identify a potent and selective LRRK2 inhibitor, LRRK2-IN-1, and demonstrated that inhibition of LRRK2 induces dephosphorylation of Ser910 and Ser935 and accumulation of LRRK2 within aggregate structures. LRRK2-IN-1 will serve as a versatile tool to pharmacologically interrogate LRRK2 biology and study its role in Parkinson's disease. © 2011 Nature America, Inc. All rights reserved.
Yang Q.,Scripps Research Institute |
Deng X.,Dana-Farber Cancer Institute |
Lu B.,Scripps Research Institute |
Cameron M.,Scripps Research Institute |
And 5 more authors.
Cancer Cell | Year: 2010
BMK1 is activated by mitogens and oncogenic signals and, thus, is strongly implicated in tumorigenesis. We found that BMK1 interacted with promyelocytic leukemia protein (PML), and inhibited its tumor-suppressor function through phosphorylation. Furthermore, activated BMK1 notably inhibited PML-dependent activation of p21. To further investigate the BMK-mediated inhibition of the tumor suppressor activity of PML in tumor cells, we developed a small-molecule inhibitor of the kinase activity of BMK1, XMD8-92. Inhibition of BMK1 by XMD8-92 blocked tumor cell proliferation in vitro and significantly inhibited tumor growth in vivo by 95%, demonstrating the efficacy and tolerability of BMK1-targeted cancer treatment in animals. © 2010 Elsevier Inc.
Johnson D.S.,Pfizer |
Stiff C.,Pfizer |
Nomanbhoy T.K.,ActivX Biosciences |
Cravatt B.F.,Scripps Research Institute |
ACS Medicinal Chemistry Letters | Year: 2011
Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (kinact/Ki and IC50 values of 40300 M-1 s-1 and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials. © 2010 American Chemical Society.
Patricelli M.P.,ActivX Biosciences |
Nomanbhoy T.K.,ActivX Biosciences |
Wu J.,ActivX Biosciences |
Brown H.,ActivX Biosciences |
And 12 more authors.
Chemistry and Biology | Year: 2011
Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading. © 2011 Elsevier Ltd.
Choi H.G.,Dana-Farber Cancer Institute |
Choi H.G.,Harvard University |
Zhang J.,University of Dundee |
Deng X.,Dana-Farber Cancer Institute |
And 9 more authors.
ACS Medicinal Chemistry Letters | Year: 2012
Activating mutations in leucine-rich repeat kinase 2 (LRRK2) are present in a subset of Parkinson's disease (PD) patients and may represent an attractive therapeutic target. Here, we report that a 2-anilino-4-methylamino-5- chloropyrimidine, HG-10-102-01 (4), is a potent and selective inhibitor of wild-type LRRK2 and the G2019S mutant. Compound 4 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3 μM in cells and is the first compound reported to be capable of inhibiting Ser910 and Ser935 phosphorylation in mouse brain following intraperitoneal delivery of doses as low as 50 mg/kg. © 2012 American Chemical Society.
PubMed | The Institute for Molecular Medicine Finland, Wellspring Biosciences, Llc, Southern Research Institute, University of Alabama at Birmingham and ActivX Biosciences
Type: Journal Article | Journal: Journal of biomolecular screening | Year: 2016
During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time-dependent signal changes and identified host kinases whose probe affinities differed significantly when compared to uninfected cells. Our screen identified 10 novel host kinases that have not been previously shown to be involved with influenza virus replication, and we validated the functional importance of these novel kinases during infection using targeted small interfering RNAs (siRNAs). The effects of kinase-targeted siRNA knockdowns on replicating virus levels were measured by quantitative reverse-transcription PCR and cytoprotection assays. We identified several novel host kinases that, when knocked down, enhanced or reduced the viral load in cell culture. This preliminary work represents the first screen of the changing host kinome in influenza virus-infected human cells.
News Article | February 22, 2017
LA JOLLA, Calif., Feb. 22, 2017 (GLOBE NEWSWIRE) -- ActivX Biosciences, Inc.®, a wholly owned subsidiary of Kyorin Pharmaceutical Co., Ltd. (Tokyo), announces the appointment of Professor Hugh Rosen of The Scripps Research Institute to the position of Chairman & President of ActivX®, effective April 1, 2017. He will succeed John W. Kozarich who has been at ActivX since 2001, serving as Chairman & President since its acquisition by Kyorin in 2004. John will stay on at ActivX as a Board Director and assume the new position of Distinguished Scientist and Executive Advisor. Professor Rosen’s 30+ year career in the pharmaceutical, biotechnology and academic sectors has been one of significant achievements. Following training in medicine in Cape Town, he received his D.Phil. as a Royal Commission for the Exhibition of 1851 Scholar at the University of Oxford. He spent 11 years at Merck Research Laboratories before becoming a Professor at TSRI (The Scripps Research Institute) in 2002. There he co-invented ozanimod and was a scientific founder of Receptos, acquired by Celgene in 2015 for $7.3 Billion, as well as BlackThorn Therapeutics, which recently closed a $40M Series A. He serves as an independent Board member at Regulus Therapeutics and will remain on the faculty of TSRI. “Hugh Rosen is a world-class translational physician/scientist and biotechnology entrepreneur,” explained Dr. Kozarich. “We are delighted that he will assume the leadership of ActivX, building on our R&D contributions to Kyorin and adding new dimensions to our cutting-edge KiNativ technology. Hugh has been a friend and colleague to me and to Kyorin for 25 years. I am honored to have him as my successor and look forward to working with him in my new role. Hugh’s appointment clearly signals Kyorin’s ongoing commitment to ActivX as a key component to their future success. This is an ideal outcome for all involved.” Dr. Rosen added that: “The opportunity to lead ActivX Biosciences is especially attractive to a physician-scientist with a record of success in drug discovery and development because the ActivX technologies have unlocked exciting and potentially transforming drug discovery opportunities. This is a tribute to the outstanding work of John Kozarich and colleagues at both ActivX and Kyorin. I look forward to continuing to work with John, his management team and Kyorin to bring significant new products forward to benefit patient outcomes, caregivers and providers. Through discovery and development, we strive to improve the public health.” Mr. Minoru Hogawa, Representative Director, President and Chief Executive Officer of Kyorin Holdings Inc., commented that: “Kyorin has been and will be creating first-in-class medicines. ActivX Biosciences is the core member for our research group activities. We believe Dr. Rosen will accelerate our research programs and accomplish our goals effectively with his wide experience.” ActivX Biosciences, Inc.® (www.activx.com ) located in La Jolla, California, is a wholly-owned subsidiary of Tokyo-based Kyorin Pharmaceutical Co., Ltd., and has drug discovery and proteomics technology capabilities. The company applies proprietary chemical technologies and high-throughput protein analysis to the drug discovery and development process. By focusing on functional proteins, ActivX® addresses disease mechanisms directly, in contrast to approaches such as expression profiling, in which the measured analyte is several steps removed from the site of drug action. ActivX and its partners utilize ActivX’s proprietary technology and profiling platform (KiNativ® - www.kinativ.com ) to address critical challenges in kinase drug discovery, including selectivity profiling of candidate drug molecules in biological samples to guide their medicinal chemistry efforts. The KiNativ platform aids in the identification of novel drug targets and biomarkers, the determination of target engagement in vivo and the characterization of off-target activities of candidate and established drugs to understand the basis of their efficacy and/or toxicity. About Kyorin Pharmaceutical Co., Ltd. Trusted among patients and professionals in the medical industry, Kyorin Pharmaceutical Co., Ltd. (http://www.kyorin-pharm.co.jp/en/), which is a core company of Kyorin Holdings Inc. (http://www.kyorin-gr.co.jp/en/), strives to be a company that contributes to the public health and is recognized as a one with social significance by improving its presence in specified therapeutic areas and through global discovery of novel drugs. Kyorin Pharmaceutical Co., Ltd. uses its franchise customer strategy in the developing and marketing ethical drugs on the core areas of respiratory, otolaryngology and urology.