Philip A Cole

Johns Hopkins University, Baltimore, Maryland, United States

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Publications (187)1654.37 Total impact

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    Beverley M Dancy, Philip A Cole
    Chemical Reviews 01/2015; DOI:10.1021/cr500452k · 45.66 Impact Factor
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    Nam Chu, Philip A Cole
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    ABSTRACT: Bruton's tyrosine kinase, an enzyme that is important for B cell function, can be activated in a number of ways.
    eLife Sciences 01/2015; 4. DOI:10.7554/eLife.07204 · 8.52 Impact Factor
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    ABSTRACT: Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, non-binding control peptides and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions, and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.
    Analytical Chemistry 10/2014; DOI:10.1021/ac503137u · 5.83 Impact Factor
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    ABSTRACT: The Type I Insulin-like Growth Factor Receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation.
    eLife Sciences 09/2014; 3. DOI:10.7554/eLife.03772 · 8.52 Impact Factor
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    ABSTRACT: S-adenosylhomocysteine hydrolase (SAHH) is an NAD+-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys-401 and Lys-408 but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys-401 and Lys-408 and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated-SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD+ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.
    Journal of Biological Chemistry 09/2014; DOI:10.1074/jbc.M114.597153 · 4.60 Impact Factor
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    ABSTRACT: S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived Capture Compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with an SAH-CC with biotin used in conjunction with streptavidin-HRP. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we use a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, 10.06 ± 2.87 μM respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent Capture Compounds.
    Analytical Biochemistry 08/2014; DOI:10.1016/j.ab.2014.08.013 · 2.31 Impact Factor
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    ABSTRACT: T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. An histone/protein acetyltransferase (HAT), p300, was recently found important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3-4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose-dependent, and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 CNS2 region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function, and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells.
    Molecular and Cellular Biology 08/2014; 34(21). DOI:10.1128/MCB.00919-14 · 5.04 Impact Factor
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    ABSTRACT: The supporting information for our paper. Includes details of all the shRNAs tested against p300 and CBP in stable cell lines, the C107 synthetic chemistry (a new compound), and the detailed enzyme kinetic characterization of our new p300 BHC (bromodomain+HAT domain+C/H3 domain) which is easily expressed and purified from insect cells (easier than HAT alone by EPL or full length).
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    ABSTRACT: We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
    Oncotarget 07/2014; 5(14):5198-5208. · 6.63 Impact Factor
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    ABSTRACT: Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging as the active sites of KDM1A-B and KDM-4A-D histone demethylases, respectively, are highly conserved. Most inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide sequence, or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation and chemical modifications. Hydrogen/deuterium exchange mass spectrometry revealed that the peptide-based inhibitors target KDM4C through substrate-independent interactions located on the surface remote from the active site within less conserved regions of KDM4C. The sites discovered in this study provide a new approach of targeting KDM4C through substrate- and cofactor-independent interactions, and may be further explored to develop potent selective inhibitors and biological probes for the KDM4 family.
    ACS Chemical Biology 07/2014; 9(9). DOI:10.1021/cb500374f · 5.36 Impact Factor
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    ABSTRACT: The invention provides inhibitors of ghrelin O-acyltransferase, and methods of making and using them. In some embodiments, the invention provides bisubstrate analog inhibitors of ghrelin O-acyltransferase, which can be effective in treating, for example, obesity and diabetes mellitus.
    Ref. No: CA2739418A1, EP2340258A2, US8772229, US20110257086, WO2010039461A2, WO2010039461A3, Year: 07/2014
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    ABSTRACT: The p300 and CBP transcriptional coactivator paralogs (p300/CBP) regulate a variety of different cellular pathways, in part, by acetylating histones and more than 70 non-histone protein substrates. Mutation, chromosomal translocation, or other aberrant activities of p300/CBP are linked to many different diseases, including cancer. Because of its pleiotropic biological roles and connection to disease, it is important to understand the mechanism of acetyl transfer by p300/CBP, in part so that inhibitors can be more rationally developed. Toward this goal, a structure of p300 bound to a Lys-CoA bisubstrate HAT inhibitor has been previously elucidated, and the enzyme’s catalytic mechanism has been investigated. Nonetheless, many questions underlying p300/CBP structure and mechanism remain. Here, we report a structural characterization of different reaction states in the p300 activity cycle. We present the structures of p300 in complex with an acetyl-CoA substrate, a CoA product, and an acetonyl-CoA inhibitor. A comparison of these structures with the previously reported p300/Lys-CoA complex demonstrates that the conformation of the enzyme active site depends on the interaction of the enzyme with the cofactor, and is not apparently influenced by protein substrate lysine binding. The p300/CoA crystals also contain two poly(ethylene glycol) moieties bound proximal to the cofactor binding site, implicating the path of protein substrate association. The structure of the p300/acetonyl-CoA complex explains the inhibitory and tight binding properties of the acetonyl-CoA toward p300. Together, these studies provide new insights into the molecular basis of acetylation by p300 and have implications for the rational development of new small molecule p300 inhibitors.
    Biochemistry 05/2014; 53(21):3415–3422. DOI:10.1021/bi500380f · 3.19 Impact Factor
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    ABSTRACT: Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.
    ACS Chemical Biology 04/2014; 9(6). DOI:10.1021/cb500018s · 5.36 Impact Factor
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    ABSTRACT: Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A2AR) has been identified as a promising target for immunotherapy, small molecule A2AR agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating non-immune pathways. To overcome these limitations, we have tethered the A2AR agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A2AR interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.
    Journal of the American Chemical Society 02/2014; 136(9). DOI:10.1021/ja5006674 · 11.44 Impact Factor
  • Zhihong Wang, Philip A Cole
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    ABSTRACT: Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been implicated in many human diseases, in particular cancer. We highlight applications of technologies and concepts derived from kinase mechanistic studies that have helped illuminate how kinases are regulated and contribute to pathophysiology.
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    ABSTRACT: FOXO1 is an important downstream mediator of the insulin signaling pathway. In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation. A reduction in nuclear FOXO1 levels then leads to suppression of hepatic glucose production. However, the mechanism leading to expression of Foxo1 gene in the fasted state is less clear. We found that Foxo1 mRNA and FOXO1 protein levels of Foxo1 were increased significantly in the liver of mice after 16h of fasting. Furthermore, Bt-cAMP stimulated the expression of Foxo1 at both mRNA and protein level in hepatocytes. Since cAMP-PKA regulates hepatic glucose production through CREB co-activators, we depleted these co-activators using adenoviral shRNAs. Interestingly, only depletion of co-activator P300 resulted in the decrease of Foxo1 mRNA and FOXO1 protein levels. In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels. By characterization of Foxo1 gene promoter, P300 regulates the Foxo1 gene expression through the binding to the tandem CRE sites in the proximal promoter region of Foxo1 gene.
    Journal of Biological Chemistry 12/2013; 289(7). DOI:10.1074/jbc.M113.540500 · 4.60 Impact Factor
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    ABSTRACT: Background:Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation.Methods:Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone.Results:The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives.Conclusion:By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.British Journal of Cancer advance online publication, 5 December 2013; doi:10.1038/bjc.2013.710
    British Journal of Cancer 12/2013; 110(1). DOI:10.1038/bjc.2013.710 · 4.82 Impact Factor
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    ABSTRACT: Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes.
    Molecular cell 11/2013; 52(3):314-24. DOI:10.1016/j.molcel.2013.10.009 · 14.46 Impact Factor
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    ABSTRACT: Ghrelin O-AcylTransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the Membrane Bound O-AcylTransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here, we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation-shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, facilitated these experiments. The MBOAT family invariant residue His338 is in the ER lumen, consistent with other family members, but conserved Asn307 is cytosolic, making it unlikely that both are involved in catalysis. Photocrosslinking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately enhance the discovery of selective inhibitors for these enzymes.
    Journal of Biological Chemistry 09/2013; DOI:10.1074/jbc.M113.510313 · 4.60 Impact Factor
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    ABSTRACT: Under physiological conditions, epidermal growth factor receptor (EGFR) tyrosine kinase activity is tightly controlled through the coordinated action of both positive and negative regulators. Aberrant EGFR activation occurs frequently in many cancer types and the endogenous EGFR feedback inhibitor, Mig6/RALT, is more efficiently phosphorylated by oncogenic EGFR variants. We have utilized expressed protein ligation to generate semisynthetic Tyr394-phosphorylated and unphosphorylated forms of the Mig6 protein and show that phosphorylation of Mig6 reduces its ability to inhibit purified, near full-length EGFR (tEGFR). We also demonstrate that the kinetic parameters of tEGFR are similar whether solubilized in detergent or reconstitutued in nanodisc bilayers. These findings suggest a mechanism by which EGFR and its family members evade negative regulation by Mig6 under pathological conditions.
    ACS Chemical Biology 09/2013; 8(11). DOI:10.1021/cb4005707 · 5.36 Impact Factor

Publication Stats

10k Citations
1,654.37 Total Impact Points


  • 2000–2015
    • Johns Hopkins University
      • • Department of Pharmacology and Molecular Sciences
      • • Department of Biology
      Baltimore, Maryland, United States
  • 2014
    • Columbia University
      • Department of Biochemistry and Molecular Biophysics
      New York City, New York, United States
  • 2006–2013
    • Johns Hopkins Medicine
      • Department of Pharmacology and Molecular Sciences
      Baltimore, Maryland, United States
    • University of Minnesota Duluth
      Duluth, Minnesota, United States
  • 2010
    • Weill Cornell Medical College
      New York, New York, United States
  • 2009
    • Regions Hospital
      Saint Paul, Minnesota, United States
  • 2008
    • Wistar Institute
      Philadelphia, Pennsylvania, United States
  • 2002
    • University of Maryland, Baltimore
      Baltimore, Maryland, United States
    • National Institute of Arthritis and Musculoskeletal and Skin Diseases
      Maryland, United States
  • 1998–2002
    • The Rockefeller University
      • Laboratory of Molecular Biophysics
      New York City, New York, United States
  • 2001
    • Cornell University
      Итак, New York, United States
  • 1997–1998
    • Harvard University
      Cambridge, Massachusetts, United States