DOCSLIB.ORG

Clofarabine Targets the Large Subunit (A) of Human Ribonucleotide Reductase in Live Cells by Assembly Into Persistent Hexamers

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Clofarabine Targets the Large Subunit (A) of Human Ribonucleotide Reductase in Live Cells by Assembly Into Persistent Hexamers View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Brief Communication Clofarabine Targets the Large Subunit (a) of Human Ribonucleotide Reductase in Live Cells by Assembly into Persistent Hexamers Yimon Aye,1 Edward J. Brignole,1,3,4 Marcus J.C. Long,5 Johnathan Chittuluru,4 Catherine L. Drennan,1,2,3 Francisco J. Asturias,4 and JoAnne Stubbe1,2,* 1Department of Chemistry 2Department of Biology 3Howard Hughes Medical Institute Massachusetts Institute of Technology, Cambridge, MA 02139, USA 4Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 5Graduate Program in Biochemistry and Biophysics, Brandeis University, Waltham, MA 02454, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2012.05.015 SUMMARY yet unresolved, active complex(es) proposed to be a2b2, a6b2, and/or a6b6 (Kashlan and Cooperman, 2003; Rofougaran et al., Clofarabine (ClF) is a drug used in the treatment 2006; Fairman et al., 2011; Hofer et al., 2012). In addition to of leukemia. One of its primary targets is human the NDP-binding, catalytic (C) site, (a2)m possesses two well- ribonucleotide reductase (hRNR), a dual-subunit, characterized allosteric sites. The activity (A) site, within the N-terminal ATP-cone-domain, binds either ATP, activating (a2)m(b2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mam- RNR activity, or dATP, inhibiting it. The specificity (S) site, at a malian cells, ClF targets hRNR by converting its the 2 interface, binds dNTP/ATP, modulating the substrate preference at the C site (Fairman et al., 2011). In vitro studies a-subunit into kinetically stable hexamers. We estab- on eukaryotic RNRs indicate that the dATP-inhibited complex lished mammalian expression platforms that enabled is a (Kashlan and Cooperman, 2003; Rofougaran et al., 2006; a 6 isolation of functional and characterization of Fairman et al., 2011; Aye and Stubbe, 2011), with its arrange- its altered oligomeric associations in response to ment as a trimer-of-dimers demonstrated by electron micros- ClF treatment. Size exclusion chromatography and copy (EM) (of particles preserved in stain) and 6.6 A˚ -resolution electron microscopy documented persistence of X-ray crystallography of the Saccharomyces cerevisiae RNR in-cell-assembled-a6. Our data validate hRNR as (Fairman et al., 2011). an important target of ClF, provide evidence that The importance of hRNR in dNTP pool homeostasis (Zhou and in vivo a’s quaternary structure can be perturbed Elledge, 2000; Thelander, 2007; Bester et al., 2011) has rendered by a nonnatural ligand, and suggest small-mole- it a successful drug target (Shao et al., 2006), and drug-induced cule-promoted, persistent hexamerization as a alterations in oligomeric equilibria have been demonstrated in vitro (Wang et al., 2007; Aye and Stubbe, 2011). We have strategy to modulate hRNR activity. These studies recently shown that di- and triphosphates of the clinically used lay foundations for documentation of RNR oligo- leukemia-drug ClF (Clofarabine, Clolar; Figure 1) are both revers- meric state within a cell. ible inhibitors of hRNR, binding to the C and A sites of a, respec- tively (Aye and Stubbe, 2011). Inactivation is accompanied by INTRODUCTION a-hexamerization that occurs independently of allosteric effec- tors and (b2)n. Size exclusion chromatography (SEC) studies Alterations of quaternary states of a protein can change its func- further demonstrated that ClFD(T)P-induced hexamers are tion dramatically (Nooren and Thornton, 2003; Arkin and Wells, kinetically stable in the absence of inhibitors in the SEC running 2004; Piehler, 2005). Although many methods exist to establish buffer. These hexamers thus display fundamentally distinct protein oligomeric equilibria in vitro, validating the existence of kinetic properties from the dATP-induced a6 (Kashlan and Coop- these oligomers and their relevance in vivo is often challenging, erman, 2003; Rofougaran et al., 2006; Fairman et al., 2011; Aye despite its importance (Piehler, 2005). One specific case where and Stubbe, 2011), which rapidly dissociate to an equilibrium these issues remain unresolved is ribonucleotide reductases mixture of lower order oligomers when the dATP is omitted (RNRs), highly regulated enzymes that catalyze the conversion from the elution buffer (Fairman et al., 2011; Aye and Stubbe, of nucleoside 50-diphosphates (NDPs) to deoxynucleotides 2011). (Stubbe and van Der Donk, 1998; Stubbe et al., 2003; Nordlund We now report biochemical and structural evidence that and Reichard, 2006). hexamers generated subsequent to RNR inhibition by ClFD(T)P Human (h)RNRs belong to the class Ia family of RNRs that in vitro (Aye and Stubbe, 2011) are also assembled in ClF-treated require two subunits (a2)m and (b2)n or p53(b2)n, which form, as cultured live cells. Initially, crosslinking studies on homogeneous Chemistry & Biology 19, 799–805, July 27, 2012 ª2012 Elsevier Ltd All rights reserved 799 Chemistry & Biology Clofarabine Induces hRNR Hexamers in Live Cells DNA Figure 1. hRNR Is a Principal Target of ClF hRNR polymerases dNTP ClF enters the cell by passive diffusion and/or via NDP dNDP DNA pools nucleoside transporters and is rapidly phosphor- ylated (Bonate et al., 2006; Zhenchuk et al., 2009). NH2 m n ClFDP and ClFTP both target hRNR (a ) , resulting N 2 m N in the depletion of dNTP pools including dCTP. N N Cl HO O Reversible inhibition Reduction of the latter potentiates production H F of ClF metabolites by removing negative feedback HO H dCK inhibition of deoxycytidine kinase, dCK, an en- ClF ClF ClFMP ClFDP ClFTP zyme that catalyzes monophosphorylation of ClF. Diminution of overall dNTP pool sizes amplifies extracellular intracellular misincorporation of ClFTP into DNA by DNA polymerase-a and –ε, where it functions as a chain terminator. Induction of apoptosis is thought to be ultimately responsible for anticancer activity. ClFTP is a rapid reversible inhibitor of a via binding to the activity (A) site (Ki = 40 nM) and ClFDP is a slow-release reversible inhibitor (Ki* = 17 nM, t1/2 = 23 min) of a via binding to the catalytic (C) site, and both cause a hex- amerization (Aye and Stubbe, 2011). Blue and green ellipses represent (a2) and (b2 or p53b2), respectively. recombinant a at concentrations measured in vivo (A˚ kerblom As a prelude to this goal, we initially attempted native gel et al., 1981; Ha˚ kansson et al., 2006; present study) showed, by electrophoresis on hexameric His6-a generated in vitro as previ- gel analysis, that in the presence of ClFD(T)P, a was almost ously described (Aye and Stubbe, 2011). However, a6 failed to exclusively hexameric. However, similar studies to demonstrate penetrate a 4% polyacrylamide gel. We then tested chemical hexamerization of the endogenous mammalian a in highly dilute crosslinking in an effort to covalently stabilize the hexameric lysates resulting from ClF-treated COS-1 cells, proved to be state for analysis by denaturing gel electrophoresis. BS-3 challenging because of very low endogenous a expression. [Bis(sulfosuccinimidyl) suberate] (Figure S1B-A) was chosen as Thus in an effort to assess the oligomeric state of a in vivo, the the crosslinker and the optimized conditions resulted in a shift protein was first overexpressed minimally (3.5-fold above the of a from 92 kDa (monomer of a) to >500 kDa (consistent with endogenous level as judged by western blot analysis). Under hexamer of a)(Figure S1B-B). Hexamerization was detected these conditions, a from ClF-treated live COS-1 cells, subse- over a range of a concentrations (5 nM to 5 mM) in the presence quent to crosslinking in the resulting lysates, underwent a gel- of BS3 (1–3 mM), subsequent to an incubation period of as early shift to a molecular weight consistent with a hexamers and as 1 min at 37C. Only when the samples had been treated with similar to that characterized in vitro (Aye and Stubbe, 2011). To ClFD(T)P and BS3 were the shifts observed. In a control with no obtain biochemical and structural data in support of a-hexame- inhibitor, or with inhibitor and no BS3, the large-molecular- rization, we then constructed a bicistronic reporter cassette that weight species was absent. In addition, crosslinked complexes simultaneously overexpressed a fluorescent protein, Discosoma with an approximate molecular weight of a dimers were also de- Red (DsRed), and His6-a (Wang et al., 2007; Aye and Stubbe, tected (to < 20% of total a) in the presence or absence of inhibitor 2011). This platform enabled assessment of the oligomeric state (Figure S1B-B). The observed gel shift thus serves as a diag- of native a by SEC and single particle EM, subsequent to cell nostic marker for a hexamerization and provided the impetus lysis and affinity purification. Our data together validate the to use crosslinking as an initial strategy to examine the ClF- current model that hRNR is a target of ClF metabolites prior to induced quaternary state perturbation of (a2)m in mammalian the onset of cytotoxicity (Bonate et al., 2006). Remarkably, cells. targeting hRNR results in the in-cell assembly of persistent Experiments on COS-1 cells treated with ClF to detect hex- hexamers that are also shown in vitro to remain stable subse- amerization of the endogenous protein proved considerably quent to inhibitor dissociation. We provide the first evidence, more challenging. Ultimately, success was achieved using a to our knowledge, that the oligomeric state of (a2)m in live cells mammalian expression plasmid (monocistronic cassette, Fig- can be perturbed by an external ligand. ure 2A and Table S1A) transfected into COS-1 cells that allowed ectopic expression of untagged hRNR a at levels 3.5-fold RESULTS above the endogenous level (Figure S1B-C).
Load more

Recommended publications
  • Genetically Encoded Fragment-Based Discovery
    Available online at www.sciencedirect.com ScienceDirect Genetically encoded fragment-based discovery 1 Ratmir Derda and Simon Ng This opinion describes recent advances of molecular discovery the fragment of interest. This opinion focuses on chemi- technology dubbed Genetically Encoded Fragment-Based cal post-translational incorporation of fragments Discovery (GE-FBD). GE-FBD starts from a known ligand or (Figure 1b). Alternatively, {FP} libraries can be built from ‘fragment’ that binds to a desired target weakly and often with unnatural amino acids (UAA) that contain fragments as low specificity. Covalent incorporation of fragment into a part of their side-chain (Figure 1c). Selection from {FP} diverse, genetically encoded library of peptides yields a library libraries has been shown to identify ligands in which the of peptide–fragment combinations. Selection from such a fragment covalently linked to peptide segment bind the library has a high likelihood to identify ligands, in which the target with improved affinity and specificity when com- peptides bind to distinct adjacent pockets of the target in pared to the original fragment (Figure 1d). Improvement F FP synergy with the fragment and exhibits enhanced affinity and can be defined as r = KD/ KD > 1 or DDGbind Àlog F specificity when compared to the fragment itself. GE-FBD (r) < 0, where KD is the affinity of the fragment towards could employ fragments that bind non-covalently as well as the target of interest (Figure 1e). Figure 1d summarizes reversible covalent warheads. The key advances in GE-FBD up-to-date outcomes from published GE-FBD reports include (i) synthetic chemistry that enables incorporation of and shows that r = 10–100 can be found across diverse F diverse fragments into both linear and cyclic peptide libraries; fragment with KD ranging from 100 nM to 1 mM [3 ,4– (ii) quantification of multi-step modifications in million-to-billion 6,7 ,8,9 ,10,11 ,12,13 ,14].
    [Show full text]
  • ICBS 2018 Vancouver, Canada
    7th Annual Conference September 24-27, 2018 ICBS 2018 Vancouver, Canada Scientific Program Towards Translational Impact www.chemical-biology.org 7th Annual Conference | September 24-27, 2018 | Vancouver, Canada Acknowledgements Towards Translational Impact September 24th – 27th, 2018 | Vancouver, BC Local Program and Organizing Committee Tom Pfeifer, Centre for Drug Research and Development Roger Linington, Simon Fraser University Michel Roberge, University of British Columbia Nicolette Honson, Centre for Drug Research and Development ICBS Organizing Committee Haian Fu, Chair, Emory University, USA Lixin Zhang, ECUST, China Sally-Ann Poulsen, Griffith University, Australia Jonathan Baell, Monash University, Australia Mahabir Gupta, University of Panama, Panama Junying Yuan, Harvard Medical School, USA Masatoshi Hagiwara, Kyoto University, Japan Petr Bartunek, CZ-OPENSCREEN and Institute of Molecular Jason Micklefield, The University of Manchester, UK Genetics, Czech Republic Siddhartha Roy, Boss Institute, India ICBS Young Chemical Biologist Award 2018 Selection Committee Yimon Aye, Cornell University, USA Christian Ottmann, Technische Universiteit Eindhoven, Ratmir Derda, University of Alberta, Canada Netherlands Evripidis Gavathiotis, Albert Einstein College of Medicine, USA William Pomerantz, University of Minnesota, USA Kenjiro Hanaoka, University of Tokyo, Japan Qi Zhang, Fudan University, China Recording of sessions (oral or poster) by audio, video, or still photography is strictly prohibited except with the advance permission of
    [Show full text]
  • California Insɵtute of Technology Division of Biology and Biological Engineering Annual Report 2017
    California InsƟtute of Technology Division of Biology and Biological Engineering Annual Report 2017 Introduction Annual Report | Biology and Biological Engineering | 2017 Introduction The annual report for Caltech's Division of Biology and Biological Engineering (BBE) presents major research accomplishments of faculty, students, and staff during the previous academic year. This report covers October 1, 2016 to September 30, 2017. Front Cover Illustration Vector-assisted spectral tracing (VAST) in the cerebellum of an adult mouse A movie highlighting the multi-color vector-assisted spectral tracing (VAST) system in the cerebellum of an adult mouse. Due to the stochastic uptake of AAV-PHP viruses encoding either a blue, green or red fluorescent protein, cells are labeled with a wide range of hues. This approach can be used to differentiate neighboring neurons for morphology and tracing studies. Credit: Ben Deverman et al., Gradinaru Lab Back Cover Illustration Engineered adeno-associated viruses efficiently cross the blood-brain-barrier for enhanced brain transduction in adult mice. Representative images of virally-delivered nuclear GFP fluorescence (green) and Calbindin immunohistochemistry (magenta) in the cerebellum. Credit: Chan et al., Gradinaru Lab 1 News, Events, and People Annual Report | Biology and Biological Engineering | 2017 Press Releases 5 Annual Retreat 14 Ferguson Prize 16 Ferguson Prize 17 Professorial Awards and Honors 18 Seminars 19 Named Lectures 25 2 News, Events, and People Annual Report | Biology and Biological Engineering
    [Show full text]
  • A Tipping Point in Cancer Research
    A Tipping Point in Cancer Research Annual Report 2011 IN THIS NEW ERA OF ACCELERATED PROGRESS, THEIR INNOVATIVE CONTRIBUTIONS HAVE it is more important than ever that we support BEEN WIDELY RECOGNIZED: creative young scientists who can harness the • 2 Damon Runyon scientists were included in A Tipping Point in Cancer Research momentum of the past decade to transform the Science magazine’s Insights of the Decade, way we prevent, detect, and treat cancer. The Celebrating Extraordinary Progress which listed “10 insights that have changed science Damon Runyon Cancer Research Foundation is since the dawn of the new millennium.” SCIENTISTS, RESEARCH ORGANIZATIONS AND THE NATIONAL CANCER INSTITUTE ALL AGREE: playing a central role by funding groundbreaking we are at a tipping point in cancer research. The convergence of knowledge from across the spectrum basic and clinical cancer research. In the past year, • 2 Damon Runyon alumni were elected to the of cutting-edge science combined with powerful new technologies has created a deeper understanding our brilliant young scientists: National Academy of Sciences (the science “Hall of Fame”), bringing Damon Runyon’s total to 61. of cancers and advanced our ability to detect and treat these diseases earlier and more effectively. • led studies resulting in FDA approval of two new drugs for advanced melanoma: Yervoy, an immu- • William R. Sellers, MD, Board Member and notherapy, and Zelboraf, a targeted therapy former Clinical Investigator, was appointed by President Obama to the National Cancer • reported the success of a novel immunotherapy Advisory Board. for pancreatic cancer • Elaine V. Fuchs, PhD, Board Member and • completed the first genome sequencing of former Fellow, received the prestigious Albany multiple myeloma and medulloblastoma Medical Center Prize.
    [Show full text]
  • Current Research in Chemical Biology
    CURRENT RESEARCH IN CHEMICAL BIOLOGY AUTHOR INFORMATION PACK TABLE OF CONTENTS XXX . • Description p.1 • Editorial Board p.2 • Guide for Authors p.3 ISSN: 2666-2469 DESCRIPTION . Current Research in Chemical Biology (CRCHBI) is a new primary research, gold open access journal from Elsevier. CRCHBI publishes original papers and short communications that cover all aspects of chemical biology and related areas. Current Research in Chemical Biology is a peer-reviewed gold open access (OA) journal and upon acceptance all articles are permanently and freely available. It is a companion to the highly regarded review journal Current Opinion in Chemical Biology and is part of the Current Opinion and Research (CO+RE) suite of journals. All CO+RE journals leverage the Current Opinion legacy- of editorial excellence, high-impact, and global reach-to ensure they are a widely read resource that is integral to scientists' workflow. Current Research in Chemical Biology topics covered include: Bioorganic and bioinorganic chemistry Biocatalysis and biotransformation (Applied) medicinal chemistry Next generation therapeutics (e.g. PROTACs, protein/peptide-based therapeutics, ..) Molecular imaging, high resolution methods in microscopy Synthetic biology and biomolecules Energy (e.g. biological pathways for electrons, protons and photo-excitations) Mechanistic biology Genome- and proteome-wide analysis Metabolomics Assay development Photo-induced processes and light switches Current Research in Chemical Biologybuilds on Elsevier's reputation for excellence in scientific publishing. Expertise - Editors and Editorial Board bring depth and breadth of expertise and experience to the journal. Speed - Submission and peer review is fast, and publication of final manuscripts is instantaneous. Discoverability - Articles get high visibility and maximum exposure on an industry-leading platform that reaches a vast global audience.
    [Show full text]
  • COURSE SCHEDULE: Biochemistry/Chemistry 945: Seminar—Chemical Biology (Advanced) Blackwell — Spring 2018 Wednesdays @ 4:35 Pm, Room 8335 Chemistry
    COURSE SCHEDULE: Biochemistry/Chemistry 945: Seminar—Chemical Biology (Advanced) Blackwell — Spring 2018 Wednesdays @ 4:35 pm, Room 8335 Chemistry Discussion Date Speaker Paper CBI-relevant Seminars Facilitator Jan Introduction Blackwell None Paula Hammond – Jan 29/30 24 Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma Jan Marc Currie patients; Wargo, Science, 2018, 359, 97–103; Herman Sintim - Jan 26 31 Chevrette Commentary: Jobin Science, 2018, 359, 32–34 Polyamine-Mediated Stoichiometric Assembly of Ribonucleoproteins for Benjamin Feb 7 Lynn Enhanced mRNA Delivery Ortiz Hammond, Angew Chem Int Ed, 2017, 56, 13709–13712 Acoustic reporter genes for noninvasive imaging of microorganisms in Feb Emily Cai mammalian hosts 14 Ehlerding Shapiro, Nature, 2018, 553, 86–90 Akt3 is a privileged first responder in isozyme-specific electrophile response Feb Mark Klein Denu Aye, Nature Chem Bio, 2017, 13, 333–338; 21 Commentary: Weerapana, Nature Chem Bio, 2017, 13, 244–245 Programmable base editing of A•T to G•C in genomic DNA without DNA Feb Rebeca Brunold cleavage Yimon Aye - Feb 27 28 Fernandez Liu, Nature, 2017, 551, 464–471 Tim Multidimensional chemical control of CRISPR-Cas9 Mar 7 Jin Tiambeng Shoulders & Choudhary, Nature Chem Bio, 2017, 13, 9–11 An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Mar Trisha Ge Cells Matt Shoulders – Mar 13 14 Tucholski Krogan, Cell, 2017, 169, 350–360 A semi-synthetic organism that stores and retrieves increased genetic Mar Joseph Blackwell information 21
    [Show full text]
  • 3.3-Å Resolution Cryo-EM Structure of Human Ribonucleotide Reductase with Substrate and Allosteric Regulators Bound
    3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Brignole, Edward J et al. “3.3-Å Resolution Cryo-EM Structure of Human Ribonucleotide Reductase with Substrate and Allosteric Regulators Bound.” eLife 7 (February 2018): e31502 © Brignole et al As Published http://dx.doi.org/10.7554/eLife.31502 Publisher eLife Sciences Publications, Ltd Version Final published version Citable link http://hdl.handle.net/1721.1/114937 Terms of Use Creative Commons Attribution 4.0 International License Detailed Terms https://creativecommons.org/licenses/by/4.0/ RESEARCH ARTICLE 3.3-A˚ resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound Edward J Brignole1,2†, Kuang-Lei Tsai3†‡, Johnathan Chittuluru3, Haoran Li4, Yimon Aye4§, Pawel A Penczek5, JoAnne Stubbe2,4*, Catherine L Drennan1,2,4*, Francisco Asturias3#* 1Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States; 2Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; 3Department of Integrative Computational and Structural Biology, The Scripps Research Institute, La Jolla, United States; *For correspondence: 4Department of Chemistry, Massachusetts Institute of Technology, Cambridge, [email protected] (JAS); 5 [email protected] (CLD); United States; Department of Biochemistry and Molecular Biology, The University [email protected] of Texas-Houston Medical School, Houston, United States (FA) †These authors contributed equally to this work Abstract Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, Present address: ‡Department a reaction essential for DNA replication and repair.
    [Show full text]
  • Table of Contents (PDF)
    June 14, 2011 u vol. 108 u no. 24 u 9727–10022 Cover image: Pictured is the ladybird beetle Delphastus catalinae next to the rim of a US one cent coin. Stephen T. Deyrup and colleagues used 2D NMR spectroscopy to screen metabolites, including defensive polyketides that these tiny beetles produce. The technique revealed partial molecular structures that allowed the authors to identify novel compounds within the complex mixtures without prior fractionation or isolation. See the article by Deyrup et al. on pages 9753–9758. Image courtesy of Frank C. Schroeder. From the Cover 9753 Searching for new chemical entities 9776 Clay minerals and carbon sequestration 9782 Innate grasp of Euclidean geometry 9851 Targeting cancer with hyperthermia 9910 Speciation among whiptail lizards 9733 Spontaneous speciation by ploidy elevation: Laboratory Contents synthesis of a new clonal vertebrate Craig Moritz and Ke Bi See companion article on page 9910 THIS WEEK IN PNAS 9727 In This Issue PNAS PLUS (AUTHOR SUMMARIES) LETTERS (ONLINE ONLY) BIOLOGICAL SCIENCES E198 Algal ice-binding proteins change the structure of sea ice CELL BIOLOGY James A. Raymond 9735 Targeting and imaging single biomolecules in living cells E199 ADAR1 isoform involvement in embryonic lethality by complementation-activated light microscopy with Richard A. Steinman and Qingde Wang split-fluorescent proteins Fabien Pinaud and Maxime Dahan E200 Reply to Steinman and Wang: Involvement of the p150 See full research article on page E201 of www.pnas.org isoform of the RNA editing enzyme ADAR1 in embryonic lethality Simone V. Ward, Cyril X. George, Charles E. Samuel, PHYSIOLOGY and Michael B.
    [Show full text]
  • Where Electrophile Signaling and Covalent Ligandâœtarget Mining
    News from New Chemistry Professors iN switzerlaNd CHIMIA 2020, 74, No. 9 659 doi:10.2533/chimia.2020.659 Chimia 74 (2020) 659–666 © Y. Aye Where Electrophile Signaling and Covalent Ligand–Target Mining Converge Yimon Aye* Abstract: Interests in learning how to engineer most effective covalent ligands, identify novel functional targets, and define precise mechanism-of-action are rapidly growing in both academia and pharmaceutical industries. We here illuminate the establishment of a multifunctional platform that offers new capabilities to logically engineer covalent ligands and dissect ‘on-target’ bioactivity with precise biological context and precision hitherto inac- cessible. Broadly aimed at non-specialist readers, this opinion piece is aimed to stoke the interest of emerging chemists and biologists/bioengineers, but the underlying technological and conceptual topicality is anticipated to also appeal to experts leading ligand–target mining, validation, and -discovery research programs. Keywords: Covalent ligands · Electrophile signalling · Ligand–Target mining Yimon Aye read chemistry at University trophilicity with respect to Group 1. Tecfidera, Vumerity,[6] and of Oxford (UK) (2000–04), and received plant-derived compounds such as curcumin and sulphoraphane her PhD degree in organic chemistry with belong to Group 2 (Fig. 1a). Generally, from reactivity–selectivity David Evans (Harvard, USA) and postdoc- principles, Group 2 compounds of magnified electrophilicity are toral training in biochemistry with JoAnne anticipated to cover a significantly broader on-/off-target spectra. Stubbe (MIT, USA), where she established the mode-of-action of therapeutics targeting When Natural and Unnatural Worlds Converge ribonucleotide reductase. In her indepen- Interestingly, Nature has endowed us with a repertoire of elec- dent career that began mid-2012, she set trophilic small-molecule metabolites having similar properties out to understand the detailed mechanisms to Group 2 molecules.
    [Show full text]
  • The Not So Identical Twins: (Dis)Similarities Between Reactive Electrophile and Oxidant Sensing and Signaling
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 14 May 2021 doi:10.20944/preprints202105.0334.v1 The not so identical twins: (dis)similarities between reactive electrophile and oxidant sensing and signaling Marcus J. C. Long‡, Kuan-Ting Huang†, and Yimon Aye†* †Swiss Federal Instute of Technology in Lausanne (EPFL), 1015 Lausanne, Switzerland ‡University of Lausanne, 1015 Lausanne, Switzerland *Correspondence: [email protected] Abstract In this tutorial review, we compare and contrast the chemical mechanisms of electrophile/oxidant sensing, and the molecular mechanisms of signal propagation. We critically analyze biological systems in which these different pathways are believed to be manifest and what the data really mean. Finally, we discuss applications of this knowledge to disease treatment and drug development. Keywords: RES; ROS; reactive chemical species; electrophile signaling; cysteine; LDE; T-REX; G-REX; Chemical Biology 1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 14 May 2021 doi:10.20944/preprints202105.0334.v1 Key learning points • Despite being promiscuous, reactive electrophilic and oxidative species (RES and ROS) show nuanced biological properties and functions driven by context-dependent covalent modification of specific proteins. • Different RES and ROS chemotypes have different interaction preferences, due to different intrinsic rates of reaction with specific functional groups. Molecules with lower intrinsic rates of reaction are most likely to be used selectively by biology. • Modulation of biological processes triggered as a result of specific interactions between specific RES/ROS and specific proteins, occurs particularly with relatively low-reactivity RES/ROS. Proteins involved in these processes typically manifest elevated reaction rates with (often specific) RES/ROS.
    [Show full text]
  • Chemical Biology Volume 48 (2019 ) Editor Benjamin G Davis UK
    Current Opinion in Chemical Biology Volume 48 ( 2019 ) Editor Benjamin G Davis UK Editorial Enquiries Editorial Board 2019 Contents Current Opinion in Chemical Biology Duilio Arigoni (Switzerland) The subject of chemical biology is divided into Elsevier Jacqueline K. Barton (USA) nine major sections, each of which is reviewed once a year. Each issue contains one or two of the Radarweg 29 Hagan Bayley (UK) sections, and the amount of space devoted to Stephen J. Benkovic (USA) 1043 NX Amsterdam each section is related to its importance. The Netherlands Carolyn Bertozzi (USA) Tel.: +31 (0)20 485 2560 Thomas Cech (USA) e-mail: [email protected] Michelle Chang (USA) February Peng Chen (China) Jason Chin (UK) Omics Benjamin F. Cravatt (USA) Edited by Ileana M. Cristea and Kathryn S. Lilley Current Opinion in Chemical Biology William F. DeGrado (USA) ISSN 1367-5931 Peter B. Dervan (USA) is published bimonthly by Alan R. Fersht (UK) April Elsevier Katharina Gaus (Australia) www.elsevier.com Biocatalysis and Biotransformation Harry B. Gray (USA) Edited by Kylie Vincent and Bettina Nestl Jay Groves (USA) The Aims of the journal can be found at Bioinorganic Chemistry Itaru Hamachi (Japan) Edited by Kyle Lancaster www.elsevier.com/locate/cbi Chuan He (USA) Paul J. Hergenrother (USA) Current Opinion in Chemical Biology Donald Hilvert (Switzerland) June is indexed and/or abstracted by Philipp Holliger (UK) BIOSIS Linda Hsieh-Wilson (USA) Next Generation Therapeutics Edited by Paul Hergenrother and Yimon Aye CAB Abstracts International Barbara Imperiali (USA) CAB Health J. Bryan Jones (Canada) Daniel Kahne (USA) Chemical Abstracts August EMBASE Stephen H.B.
    [Show full text]
  • 3.3-A˚Resolution Cryo-EM Structure of Human Ribonucleotide Reductase
    RESEARCH ARTICLE 3.3-A˚ resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound Edward J Brignole1,2†, Kuang-Lei Tsai3†‡, Johnathan Chittuluru3, Haoran Li4, Yimon Aye4§, Pawel A Penczek5, JoAnne Stubbe2,4*, Catherine L Drennan1,2,4*, Francisco Asturias3#* 1Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States; 2Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; 3Department of Integrative Computational and Structural Biology, The Scripps Research Institute, La Jolla, United States; *For correspondence: 4Department of Chemistry, Massachusetts Institute of Technology, Cambridge, [email protected] (JAS); 5 [email protected] (CLD); United States; Department of Biochemistry and Molecular Biology, The University [email protected] of Texas-Houston Medical School, Houston, United States (FA) †These authors contributed equally to this work Abstract Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, Present address: ‡Department a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, of Biochemistry and Molecular the a subunit contains the active site, and the b subunit houses the radical cofactor. Here, we Biology, The University of Texas- present a 3.3-A˚ resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of Houston Medical School, human RNR. This structure, which was determined in the presence of substrate CDP and allosteric Houston, United States; regulators ATP and dATP, has three a units arranged in an a ring. At near-atomic resolution, §Department of Chemistry & 2 6 these data provide insight into the molecular basis for CDP recognition by allosteric specificity Chemical Biology, Cornell University, Ithaca, United States; effectors dATP/ATP.
    [Show full text]