I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.
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Jun 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: The worldwide public health and socioeconomic consequences caused by the COVID-19 pandemic highlight the importance of increasing preparedness for viral disease outbreaks by providing rapid disease prevention and treatment strategies. The NSP3 macrodomain of coronaviruses including SARS-CoV-2 is among the viral protein repertoire that was identified as a potential target for the development of antiviral agents, due to its critical role in viral replication and consequent pathogenicity in the host. By combining virtual and biophysical screening efforts, we discovered several experimental small molecules and FDA-approved drugs as inhibitors of the NSP3 macrodomain. Analogue characterisation of the hit matter and crystallographic studies confirming binding modes, including that of the antibiotic compound aztreonam, to the active site of the macrodomain provide valuable structure–activity relationship information that support current approaches and open up new avenues for NSP3 macrodomain inhibitor development.
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Feb 2023
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Stefan
Gahbauer
,
Galen J.
Correy
,
Marion
Schuller
,
Matteo P.
Ferla
,
Yagmur Umay
Doruk
,
Moira
Rachman
,
Taiasean
Wu
,
Morgan
Diolaiti
,
Siyi
Wang
,
R. Jeffrey
Neitz
,
Daren
Fearon
,
Dmytro S.
Radchenko
,
Yurii S.
Moroz
,
John J.
Irwin
,
Adam R.
Renslo
,
Jenny C.
Taylor
,
Jason E.
Gestwicki
,
Frank
Von Delft
,
Alan
Ashworth
,
Ivan
Ahel
,
Brian K.
Shoichet
,
James S.
Fraser
Open Access
Abstract: The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.
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Jan 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2,3,4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6,7,8,9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT–DarG toxin–antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP–HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT–DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.
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Aug 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Dávid
Bajusz
,
Warren S.
Wade
,
Grzegorz
Satała
,
Andrzej J.
Bojarski
,
Janez
Ilaš
,
Jessica
Ebner
,
Florian
Grebien
,
Henrietta
Papp
,
Ferenc
Jakab
,
Alice
Douangamath
,
Daren
Fearon
,
Frank
Von Delft
,
Marion
Schuller
,
Ivan
Ahel
,
Amanda
Wakefield
,
Sándor
Vajda
,
János
Gerencsér
,
Péter
Pallai
,
György M.
Keserű
Diamond Proposal Number(s):
[27001, 18145, 27963]
Open Access
Abstract: Fragment-based drug design has introduced a bottom-up process for drug development, with improved sampling of chemical space and increased effectiveness in early drug discovery. Here, we combine the use of pharmacophores, the most general concept of representing drug-target interactions with the theory of protein hotspots, to develop a design protocol for fragment libraries. The SpotXplorer approach compiles small fragment libraries that maximize the coverage of experimentally confirmed binding pharmacophores at the most preferred hotspots. The efficiency of this approach is demonstrated with a pilot library of 96 fragment-sized compounds (SpotXplorer0) that is validated on popular target classes and emerging drug targets. Biochemical screening against a set of GPCRs and proteases retrieves compounds containing an average of 70% of known pharmacophores for these targets. More importantly, SpotXplorer0 screening identifies confirmed hits against recently established challenging targets such as the histone methyltransferase SETD2, the main protease (3CLPro) and the NSP3 macrodomain of SARS-CoV-2.
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May 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Marion
Schuller
,
Galen J.
Correy
,
Stefan
Gahbauer
,
Daren
Fearon
,
Taiasean
Wu
,
Roberto Efraín
Díaz
,
Iris D.
Young
,
Luan
Carvalho Martins
,
Dominique H.
Smith
,
Ursula
Schulze-Gahmen
,
Tristan W.
Owens
,
Ishan
Deshpande
,
Gregory E.
Merz
,
Aye C.
Thwin
,
Justin T.
Biel
,
Jessica K.
Peters
,
Michelle
Moritz
,
Nadia
Herrera
,
Huong T.
Kratochvil
,
Anthony
Aimon
,
James
Bennett
,
Jose
Brandao Neto
,
Aina E.
Cohen
,
Alexandre
Dias
,
Alice
Douangamath
,
Louise
Dunnett
,
Oleg
Fedorov
,
Matteo P.
Ferla
,
Martin R.
Fuchs
,
Tyler J.
Gorrie-Stone
,
James M.
Holton
,
Michael G.
Johnson
,
Tobias
Krojer
,
George
Meigs
,
Alisa J.
Powell
,
Johannes Gregor Matthias
Rack
,
Victor
Rangel
,
Silvia
Russi
,
Rachael E.
Skyner
,
Clyde A.
Smith
,
Alexei S.
Soares
,
Jennifer L.
Wierman
,
Kang
Zhu
,
Peter
O’brien
,
Natalia
Jura
,
Alan
Ashworth
,
John J.
Irwin
,
Michael C.
Thompson
,
Jason E.
Gestwicki
,
Frank
Von Delft
,
Brian K.
Shoichet
,
James S.
Fraser
,
Ivan
Ahel
Diamond Proposal Number(s):
[27001]
Open Access
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
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Apr 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[27001]
Open Access
Abstract: Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.
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Nov 2020
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I02-Macromolecular Crystallography
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Marion
Schuller
,
Kerstin
Riedel
,
Ian
Gibbs-Seymour
,
Kristin
Uth
,
Christian
Sieg
,
André P
Gehring
,
Ivan
Ahel
,
Franz
Bracher
,
Benedikt M.
Kessler
,
Jonathan M.
Elkins
,
Stefan
Knapp
Abstract: Macrodomains are conserved protein interaction modules that can be found in all domains of life as well as in certain viruses. Macrodomains mediate recognition of sequence motifs harbouring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterisation of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallisation of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localisation to sites of DNA damage.
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Oct 2017
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Sean
Froese
,
Jolanta
Kopec
,
Fiona
Fitzpatrick
,
Marion
Schuller
,
Thomas
Mccorvie
,
Rod
Chalk
,
Tanja
Plessl
,
Victoria
Fettelschoss
,
Brian
Fowler
,
Matthias R.
Baumgartner
,
Wyatt
Yue
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Background: Two intracellular proteins,
MMACHC and MMADHC, functionally interact
for cobalamin trafficking.
Results: MMADHC crystal structure reveals
protein-interacting regions and unexpected
homology to MMACHC; mutations on either
protein interfere with complex formation via
different mechanisms.
Conclusion: Complex formation likely depends on
prior cobalamin processing and can be broken by
disease mutations.
Significance: MMACHC-MMADHC
heterodimerization forms the essential trafficking
chaperone delivering cobalamin to client enzymes.
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Oct 2015
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