Development of Accessible Peptidic Tool Compounds To Study the Phosphatase PTP1B in Intact Cells.
Meyer, C., Hoeger, B., Temmerman, K., Tatarek-Nossol, M., Pogenberg, V., Bernhagen, J., Wilmanns, M., Kapurniotu, A. & Köhn, M.
ACS Chem Biol. 2014 Mar 21;9(3):769-76. doi: 10.1021/cb400903u. Epub 2014 Jan 15.
Protein tyrosine phosphatases (PTPs) play crucial roles in health and disease. Chemical modulators of their activity are vital tools to study their function. An important aspect is the accessibility of these tools, which is usually limited or not existent due to the required, often complex synthesis of the molecules. We describe here a strategy for the development of cellular active inhibitors and in-cell detection tools for PTP1B as a model PTP, which plays important roles in diabetes, obesity, and cancer. The tool compounds are based on a peptide sequence from PTP1B's substrate Src, and the resulting compounds are commercially accessible through standard peptide synthesis. The peptide inhibitor is remarkably selective against a panel of PTPs. We provide the co-crystal structure of PTP1B with the sequence from Src and the optimized peptide inhibitor, showing the molecular basis of the interaction of PTP1B with part of its natural substrate and explaining the crucial interactions to enhance binding affinity, which are made possible by simple optimization of the sequence. Our approach enables the broad accessibility of PTP1B tools to researchers and has the potential for the systematic development of accessible PTP modulators to enable the study of PTPs.
Dual-Specificity Phosphatases as Molecular Targets for Inhibition in Human Disease.
Rios, P., Nunes-Xavier, C.E., Tabernero, L., Köhn, M. & Pulido, R.
Antioxid Redox Signal. 2014 Mar 11.
Abstract Significance: The dual-specificity phosphatases (DUSPs) constitute a heterogeneous group of cysteine-based protein tyrosine phosphatases, whose members exert a pivotal role in cell physiology by dephosphorylation of phosphoserine, phosphothreonine, and phosphotyrosine residues from proteins, as well as other non-proteinaceous substrates. Recent Advances: A picture is emerging in which a selected group of DUSP enzymes display overexpression or hyperactivity that is associated with human disease, especially human cancer, making feasible targeted therapy approaches based on their inhibition. A panoply of molecular and functional studies on DUSPs have been performed in the previous years, and drug-discovery efforts are ongoing to develop specific and efficient DUSP enzyme inhibitors. This review summarizes the current status on inhibitory compounds targeting DUSPs that belong to the MAP kinase phosphatases-, small-sized atypical-, and phosphatases of regenerating liver subfamilies, whose inhibition could be beneficial for the prevention or mitigation of human disease. Critical Issues: Achieving specificity, potency, and bioavailability are the major challenges in the discovery of DUSP inhibitors for the clinics. Clinical validation of compounds or alternative inhibitory strategies of DUSP inhibition has yet to come. Future Directions: Further work is required to understand the dual role of many DUSPs in human cancer, their function-structure properties, and to identify their physiologic substrates. This will help in the implementation of therapies based on DUSPs inhibition. Antioxid. Redox Signal. 00: 000-000.
Chemical activators of protein phosphatase-1 induce calcium release inside intact cells.
Reither, G., Chatterjee, J., Beullens, M., Bollen, M., Schultz, C. & Köhn, M.
Chem Biol. 2013 Sep 19;20(9):1179-86. doi: 10.1016/j.chembiol.2013.07.008. Epub2013 Aug 22.
Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase that is involved in numerous cellular processes. PP1-disrupting peptides (PDPs) are selective chemical tools used to study PP1. They generate catalytically active PP1 inside cells but do not bind to the closely related PP2A. Here, we show that PDPs also do not act directly on PP2B, thus demonstrating the selectivity of PDPs toward PP1. We present PDPs with different properties, enabling reversible versus permanent activation of PP1. We also show that Ca(2+) spiking is an acute effect caused by PDP-induced activation of PP1. The Ca(2+) is released from internal stores. Our data show that PDPs can be used as selective chemical genetics tools to study acute and long-term effects of PP1 activation in intact cells, and PDPs will therefore be valuable tools to study PP1 biology.
Targeting the untargetable: recent advances in the selective chemical modulation of protein phosphatase-1 activity.
Chatterjee, J. & Köhn, M.
Curr Opin Chem Biol. 2013 Jun;17(3):361-8. doi: 10.1016/j.cbpa.2013.04.008. Epub2013 May 3.
Protein phosphatase-1 (PP1) has long been neglected as a potential drug target owing to its misinterpreted unselective nature. However, growing evidence demonstrates that PP1 is highly selective in complex with regulatory proteins at the holoenzyme level, each of which is involved in different essential cellular signaling events. Here we summarize promising approaches to specifically activate or inhibit PP1 activity, and discuss remaining challenges and potential solutions. The summarized chemical tools pave the way for a better understanding of PP1's role in signaling networks, and the effects resulting from their application suggest their potential as future therapeutic candidates.
Elucidating human phosphatase-substrate networks.
Li, X., Wilmanns, M., Thornton, J. & Köhn, M.
Sci Signal. 2013 May 14;6(275):rs10. doi: 10.1126/scisignal.2003203.
Phosphatases are crucially involved in cellular processes by dephosphorylating cellular components. We describe a structure-based classification scheme for all active human phosphatases that reveals previously unrecognized relationships between them. By collating protein and nonprotein substrates and integrating colocalization and coexpression data, we generated a human phosphatase-substrate network. Analysis of the protein sequences surrounding sites of dephosphorylation suggested that common recognition mechanisms may apply to both kinases and a subset of phosphatases. Analysis of three-dimensional substrate recognition by protein phosphatases revealed preferred domains in the substrates. We identified phosphatases with highly specific substrates and those with less specificity by examining the relationship between phosphatases, kinases, and their shared substrates and showed how this analysis can be used to generate testable hypotheses about phosphatase biological function. DEPOD (human DEPhOsphorylation Database, version 1.0, http://www.DEPOD.org) is an online resource with information about active human phosphatases, their substrates, and the pathways in which they function. The database includes links to kinases and chemical modulators of phosphatase activity and contains a sequence similarity search function for identifying related proteins in other species.
Challenges and opportunities in the development of protein phosphatase-directed therapeutics.
De Munter, S., Kohn, M. & Bollen, M.
ACS Chem Biol. 2013 Jan 18;8(1):36-45. doi: 10.1021/cb300597g. Epub 2012 Dec 20.
Protein phosphatases have both protective and promoting roles in the etiology of diseases. A prominent example is the existence of oncogenic as well as tumor-suppressing protein phosphatases. A few protein phosphatase activity modulators are already applied in therapies. These were however not developed in target-directed approaches, and the recent discovery of phosphatase involvement followed their application in therapy. Nevertheless, these examples demonstrate that small molecules can be generated that modulate the activity of protein phosphatases and are beneficial for the treatment of protein phosphorylation diseases. We describe here strategies for the development of activators and inhibitors of protein phosphatases and clarify some long-standing misconceptions concerning the druggability of these enzymes. Recent developments suggest that it is feasible to design potent and selective protein phosphatase modulators with a therapeutic potential.
Molecular mechanisms of the PRL phosphatases.
Rios, P., Li, X. & Köhn, M.
FEBS J. 2013 Jan;280(2):505-24. doi: 10.1111/j.1742-4658.2012.08565.x. Epub 2012Apr 10.
The phosphatases of regenerating liver (PRLs) are an intriguing family of dual specificity phosphatases due to their oncogenicity. The three members are small, single domain enzymes. We provide an overview of the phosphatases of regenerating liver, compare them to related phosphatases, and review recent reports about each phosphatase. Finally, we discuss similarities and differences between the phosphatases of regenerating liver, focusing on their molecular mechanisms and signalling pathways.
Development of a peptide that selectively activates protein phosphatase-1 in living cells.
Chatterjee, J., Beullens, M., Sukackaite, R., Qian, J., Lesage, B., Hart, D.J., Bollen, M. & Köhn, M.
Angew Chem Int Ed Engl. 2012 Oct 1;51(40):10054-9. doi: 10.1002/anie.201204308.Epub 2012 Sep 7. Europe PMC
A Molecular Tete-a-Tete Arranged by a Designed Adaptor Protein.
Meyer, C. & Köhn, M.
Angew Chem Int Ed Engl. 2012 Aug 13;51(33):8160-2. doi: 10.1002/anie.201203345.Epub 2012 Jul 13.
Come together: The joining of two miniature proteins binding different protein targets in a synthetic adaptor protein is a novel way to induce proximity between proteins. The formation of a ternary complex was shown to cause the phosphorylation of a noninherent substrate (hDM2) by the kinase Hck. The approach holds promise to become a genetically encodable system to redirect enzyme activities in vivo.
Chemical biology techniques unlock the secrets of casein kinase 2 regulation by phosphorylation and glycosylation.
Pavic, K. & Köhn, M.
Chembiochem. 2012 Jun 18;13(9):1253-5. doi: 10.1002/cbic.201200235. Epub 2012 May3.
The key to understanding: The application of expressed protein ligation and protein microarrays enabled an unparalleled insight into the complex interaction of phosphorylation and glycosylation on casein kinase 2 and its biological outcome.
Omics and chemical biology-a powerful synergism.
Curr Opin Chem Biol. 2012 Apr;16(1-2):204-5. Epub 2012 Mar 23. Europe PMC
Development of a solid phase synthesis strategy for soluble phosphoinositide analogues.
Bru, M., Kotkar, S.P., Kar, N. & Köhn, M.
Chem. Sci., 2012, 3, 1893-1902, doi: 10.1039/C2SC01061E.
Phosphatidyl inositol phosphates (PIPn) exist in nature with a variety of phosphorylation patterns on the inositol head group. These compounds are vital signaling molecules that regulate numerous key cellular processes. Different enzymes such as kinases and phosphatases modify the phosphorylation patterns. There are only a few structure activity relationship (SAR) studies with PIPn analogues to gather information for the development of PIPn analogue-based inhibitors, because chemical modifications of the inositol head group are very challenging and laborious due to multiple synthetic steps that often require tedious purifications. To simplify such studies, we describe here the development of the first solid phase organic synthesis (SPOS) strategy for PIPn analogues and derivatives providing the basis for the modification of the inositol head group in a combinatorial fashion. Our strategy builds for the first time on only four inositol building blocks to address the seven phosphorylation patterns, and this is enabled by a novel selective benzylidene acetal ring opening on a solid support, which is not possible in solution so far. The first structure?activity-relationship studies using PI(4,5)P2 analogues and derivatives with lipid phosphatases are presented.
The Metastasis-Promoting Phosphatase PRL-3 Shows Activity toward Phosphoinositides.
McParland, V., Varsano, G., Li, X., Thornton, J., Baby, J., Aravind, A., Meyer, C., Pavic, K., Rios, P. & Köhn, M.
Biochemistry. 2011 Sep 6;50(35):7579-7590. Epub 2011 Aug 11.
Phosphatase of regenerating liver 3 (PRL-3) is suggested as a biomarker and therapeutic target in several cancers. It has a well-established causative role in cancer metastasis. However, little is known about its natural substrates, pathways, and biological functions, and only a few protein substrates have been suggested so far. To improve our understanding of the substrate specificity and molecular determinants of PRL-3 activity, the wild-type (WT) protein, two supposedly catalytically inactive mutants D72A and C104S, and the reported hyperactive mutant A111S were tested in vitro for substrate specificity and activity toward phosphopeptides and phosphoinositides (PIPs), their structural stability, and their ability to promote cell migration using stable HEK293 cell lines. We discovered that WT PRL-3 does not dephosphorylate the tested phosphopeptides in vitro. However, as shown by two complementary biochemical assays, PRL-3 is active toward the phosphoinositide PI(4,5)P(2). Our experimental results substantiated by molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. The C104S variant was shown to be not only catalytically inactive but also structurally destabilized and unable to promote cell migration, whereas WT PRL-3 promotes cell migration. The D72A mutant is structurally stable and does not dephosphorylate the unnatural substrate 3-O-methylfluorescein phosphate (OMFP). However, we observed residual in vitro activity of D72A against PI(4,5)P(2), and in accordance with this, it exhibits the same cellular phenotype as WT PRL-3. Our analysis of the A111S variant shows that the hyperactivity toward the unnatural OMFP substrate is not apparent in dephosphorylation assays with phosphoinositides: the mutant is completely inactive against PIPs. We observed significant structural destabilization of this variant. The cellular phenotype of this mutant equals that of the catalytically inactive C104S mutant. These results provide a possible explanation for the absence of the conserved Ser of the PTP catalytic motif in the PRL family. The correlation of the phosphatase activity toward PI(4,5)P(2) with the observed phenotypes for WT PRL-3 and the mutants suggests a link between the PI(4,5)P(2) dephosphorylation by PRL-3 and its role in cell migration.
Efficient Scaled-Up Synthesis of N-a-Fmoc 4-Phosphono(difluoromethyl)-Lphenylalanine and Its Incorporation into Peptides
Meyer, C. and Köhn, M.
Synthesis 2011 Oct 17;20:3255-3260. Epub 2011 Sep 6.
A rapid, robust and efficient scaled-up synthetic strategy for N-a-Fmoc-4-phosphono(difluoromethyl)-L-phenylalanine and its direct incorporation into peptides is presented herein.
Preparation of biomolecule microstructures and microarrays by thiol-ene photoimmobilization.
Weinrich, D., Köhn, M., Jonkheijm, P., Westerlind, U., Dehmelt, L., Engelkamp, H., Christianen, P.C., Kuhlmann, J., Maan, J.C., Nusse, D., Schroder, H., Wacker, R., Voges, E., Breinbauer, R., Kunz, H., Niemeyer, C.M. & Waldmann, H.
Chembiochem. 2010 Jan 25;11(2):235-47.
A mild, fast and flexible method for photoimmobilization of biomolecules based on the light-initiated thiol-ene reaction has been developed. After investigation and optimization of various surface materials, surface chemistries and reaction parameters, microstructures and microarrays of biotin, oligonucleotides, peptides, and MUC1 tandem repeat glycopeptides were prepared with this photoimmobilization method. Furthermore, MUC1 tandem repeat glycopeptide microarrays were successfully used to probe antibodies in mouse serum obtained from vaccinated mice. Dimensions of biomolecule microstructures were shown to be freely controllable through photolithographic techniques, and features down to 5 microm in size covering an area of up to 75x25 mm were created. Use of a confocal laser microscope with a UV laser as UV-light source enabled further reduction of biotin feature size opening access to nanostructured biochips.
Structure-activity analysis of semisynthetic nucleosomes: mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B.
McGinty, R.K., Köhn, M., Chatterjee, C., Chiang, K.P., Pratt, M.R. & Muir, T.W.
ACS Chem Biol. 2009 Nov 20;4(11):958-68.
Post-translational modification of histones plays an integral role in regulation of genomic expression through modulation of chromatin structure and function. Chemical preparations of histones bearing these modifications allows for comprehensive in vitro mechanistic investigation into their action to deconvolute observations from genome-wide studies in vivo. Previously, we reported the semisynthesis of ubiquitylated histone H2B (uH2B) using two orthogonal expressed protein ligation reactions. Semisynthetic uH2B, when incorporated into nucleosomes, directly stimulates methylation of histone H3 lysine 79 (K79) by the methyltransferase, disruptor of telomeric silencing-like (Dot1L). Although recruitment of Dot1L to the nucleosomal surface by uH2B could be excluded, comprehensive mechanistic analysis was precluded by systematic limitations in the ability to generate uH2B in large scale. Here we report a highly optimized synthesis of ubiquitylated H2B bearing a G76A point mutation u(G76A)H2B, yielding tens of milligrams of ubiquitylated protein. u(G76A)H2B is indistinguishable from the native uH2B by Dot1L, allowing for detailed studies of the resultant trans-histone crosstalk. Kinetic and structure-activity relationship analyses using u(G76A)H2B suggest a noncanonical role for ubiquitin in the enhancement of the chemical step of H3K79 methylation. Furthermore, titration of the level of uH2B within the nucleosome revealed a 1:1 stoichiometry of Dot1L activation.
Immobilization strategies for small molecule, peptide and protein microarrays.
J Pept Sci. 2009 Mar 23;15(6):393-397.
Protein, peptide and small molecule microarrays are valuable tools in biological research. In the last decade, substantial progress has been achieved to make these powerful technologies more reliable and available for researchers. This review describes chemical preparation methods for these microarrays with focus on site-selective and bioorthogonal immobilization reactions, particularly the Staudinger ligation and the thiol-ene reaction. In addition, the application of peptide microarrays, which were prepared by Staudinger ligation, to substrate specificity mapping is illustrated. Copyright (c) 2009 European Peptide Society and John Wiley & Sons, Ltd.
Chemical biologists gather in Heidelberg.
Köhn, M. & Schultz, C.
Nat Chem Biol. 2009 Feb;5(2):66-9.
Chemical biology is well defined at its core--chemistry helping to answer biological questions--yet the boundaries are rather fuzzy. What are the differences between chemical biology and pharmacology? Is intracellular imaging a branch of chemical biology, and what about screening libraries? At Chemical Biology 2008, held in Heidelberg in October, participants heard presentations covering all these topics and more.
Simultaneous protein tagging in two colors.
Schultz, C. & Köhn, M.
Chem Biol. 2008 Feb;15(2):91-2.
The fluorescent tagging of proteins in the natural environment of the cell is an emerging technique in cell biology. In this issue of Chemistry & Biology, Gautier et al. introduce a fluorescent labeling procedure orthogonal to existing ones, enabling tagging of two different proteins in living cells.
Photochemical surface patterning by the thiol-ene reaction.
Jonkheijm, P., Weinrich, D., Köhn, M., Engelkamp, H., Christianen, P.C., Kuhlmann, J., Maan, J.C., Nusse, D., Schroeder, H., Wacker, R., Breinbauer, R., Niemeyer, C.M. & Waldmann, H.
Angew Chem Int Ed Engl. 2008;47(23):4421-4. Europe PMC
A microarray strategy for mapping the substrate specificity of protein tyrosine phosphatase.
Köhn, M., Gutierrez-Rodriguez, M., Jonkheijm, P., Wetzel, S., Wacker, R., Schroeder, H., Prinz, H., Niemeyer, C.M., Breinbauer, R., Szedlacsek, S.E. & Waldmann, H.
Angew Chem Int Ed Engl. 2007;46(40):7700-3. Europe PMC
Site-selective protein immobilization by Staudinger ligation.
Watzke, A., Köhn, M., Gutierrez-Rodriguez, M., Wacker, R., Schroder, H., Breinbauer, R., Kuhlmann, J., Alexandrov, K., Niemeyer, C.M., Goody, R.S. & Waldmann, H.
Angew Chem Int Ed Engl. 2006 Feb 20;45(9):1408-12. Europe PMC
Diels-Alder ligation and surface immobilization of proteins.
de Araujo, A.D., Palomo, J.M., Cramer, J., Köhn, M., Schroder, H., Wacker, R., Niemeyer, C., Alexandrov, K. & Waldmann, H.
Angew Chem Int Ed Engl. 2005 Dec 23;45(2):296-301. Europe PMC
Functional evaluation of carbohydrate-centred glycoclusters by enzyme-linked lectin assay: ligands for concanavalin A.
Köhn, M., Benito, J.M., Ortiz Mellet, C., Lindhorst, T.K. & Garcia Fernandez, J.M.
Chembiochem. 2004 Jun 7;5(6):771-7.
The affinities of the mannose-specific lectin concanavalin A (Con A) towards D-glucose-centred mannosyl clusters differing in the anomeric configuration of the monosaccharide core, nature of the bridging functional groups and valency, have been measured by a competitive enzyme-linked lectin assay. Pentavalent thioether-linked ligands (5 and 7) were prepared by radical addition of 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranose to the corresponding penta-O-allyl-alpha- or -beta-D-glucopyranose, followed by deacetylation. The distinct reactivity of the anomeric position in the D-glucose scaffold was exploited in the preparation of a tetravalent cluster (10) that keeps a reactive aglyconic group for further manipulation, including incorporation of a reporter group or attachment to a solid support. Hydroboration of the double bonds in the penta-O-allyl-alpha-D-glucopyranose derivative and replacement of the hydroxy groups with amine moieties gave a suitable precursor for the preparation of pentavalent and 15-valent mannosides through the thiourea-bridging reaction (17 and 20, respectively). The diastereomeric 1-thiomannose-coated clusters 5 and 7 were demonstrated to be potent ligands for Con A, with IC(50) values for the inhibition of the Con A-yeast mannan association indicative of 6.4- and 5.5-fold increases in binding affinity (valency-corrected values), respectively, relative to the value for methyl alpha-D-mannopyranoside. The tetravalent cluster 10 exhibited a valency-corrected relative lectin-binding potency virtually identical to that of the homologous pentavalent mannoside 7. In sharp contrast, replacement of the 1-thiomannose wedges of 5 with alpha-D-mannopyranosylthioureido units (17) virtually abolished any multivalent or statistic effects, with a dramatic decrease of binding affinity. The 15-valent ligand 20, possessing classical O-glycosidic linkages, exhibited a twofold increase in lectin affinity relative to the penta-O-(thioglycoside) 5; it is less efficient based on the number of mannose units. The results illustrate the potential of carbohydrates as polyfunctional platforms for glycocluster construction and underline the importance of careful design of the overall architecture in optimising glycocluster recognition by specific lectins.
The Staudinger ligation-a gift to chemical biology.
Köhn, M. & Breinbauer, R.
Angew Chem Int Ed Engl. 2004 Jun 14;43(24):3106-16.
Although the reaction between an azide and a phosphane to form an aza-ylide was discovered by Hermann Staudinger more than 80 years ago and has found widespread application in organic synthesis, its potential as a highly chemoselective ligation method for the preparation of bioconjugates has been recognized only recently. As the two reaction partners are bioorthogonal to almost all functionalities that exist in biological systems and react at room temperature in an aqueous environment, the Staudinger ligation has even found application in the complex environment of living cells. Herein we describe the current state of knowledge on this reaction and its application both for the preparation of bioconjugates and as a ligation method in chemical biology.
Small Molecule Arrays.
Breinbauer, R., Köhn, M. & Peters, C.
in C. Schmuck, H. Wennemers (Eds.), 2004, Highlights in Bioorganic Chemistry, Wiley-VCH, Weinheim
Azide-alkyne coupling: a powerful reaction for bioconjugate chemistry.
Breinbauer, R. & Köhn, M.
Chembiochem. 2003 Nov 7;4(11):1147-9. Europe PMC
Staudinger ligation: a new immobilization strategy for the preparation of small-molecule arrays.
Köhn, M., Wacker, R., Peters, C., Schroder, H., Soulere, L., Breinbauer, R., Niemeyer, C.M. & Waldmann, H.
Angew Chem Int Ed Engl. 2003;42(47):5830-4. Europe PMC