Publications by authors named "Lluís Raich"

22 Publications

  • Page 1 of 1

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

EBioMedicine 2021 Mar 4;65:103255. Epub 2021 Mar 4.

Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, 37077 Göttingen, Germany; Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany. Electronic address:

Background: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated.

Methods: We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA.

Findings: We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity.

Interpretation: Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA.

Funding: NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.
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http://dx.doi.org/10.1016/j.ebiom.2021.103255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930809PMC
March 2021

Discovery of a hidden transient state in all bromodomain families.

Proc Natl Acad Sci U S A 2021 Jan;118(4)

Department of Mathematics and Computer Science, Freie Universität Berlin, 14195 Berlin, Germany;

Bromodomains (BDs) are small protein modules that interact with acetylated marks in histones. These posttranslational modifications are pivotal to regulate gene expression, making BDs promising targets to treat several diseases. While the general structure of BDs is well known, their dynamical features and their interplay with other macromolecules are poorly understood, hampering the rational design of potent and selective inhibitors. Here, we combine extensive molecular dynamics simulations, Markov state modeling, and available structural data to reveal a transiently formed state that is conserved across all BD families. It involves the breaking of two backbone hydrogen bonds that anchor the ZA-loop with the α helix, opening a cryptic pocket that partially occludes the one associated to histone binding. By analyzing more than 1,900 experimental structures, we unveil just two adopting the hidden state, explaining why it has been previously unnoticed and providing direct structural evidence for its existence. Our results suggest that this state is an allosteric regulatory switch for BDs, potentially related to a recently unveiled BD-DNA-binding mode.
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http://dx.doi.org/10.1073/pnas.2017427118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848705PMC
January 2021

Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2-Priming Protease TMPRSS2.

bioRxiv 2020 Oct 7. Epub 2020 Oct 7.

Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. The main cellular location where the SARS-CoV-2 S protein priming occurs remains debatable, therefore hampering the development of targeted treatments. Herein, we identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease-inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Our data support the key role of extracellular serine proteases in SARS-CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.

Summary: Delivery of extracellular serine protease inhibitors (serpins) such as A1AT has the capacity to reduce SARS-CoV-2 dissemination by binding and inhibiting extracellular proteases on the host cells, thus, inhibiting the first step in SARS-CoV-2 cell cycle (i.e. cell entry).
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http://dx.doi.org/10.1101/2020.05.04.077826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553163PMC
October 2020

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

bioRxiv 2020 Aug 5. Epub 2020 Aug 5.

Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, 37077 Göttingen, Germany.

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.
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http://dx.doi.org/10.1101/2020.08.05.237651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418737PMC
August 2020

An Epoxide Intermediate in Glycosidase Catalysis.

ACS Cent Sci 2020 May 16;6(5):760-770. Epub 2020 Apr 16.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 -α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope () conformation. Kinetic isotope effects (/) for anomeric-H and anomeric-C support an oxocarbenium ion-like transition state, and that for C2-O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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http://dx.doi.org/10.1021/acscentsci.0c00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256955PMC
May 2020

A Single Point Mutation Converts GH84 -GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence.

J Am Chem Soc 2020 02 24;142(5):2120-2124. Epub 2020 Jan 24.

Departament de Quı́mica Inorgànica i Orgànica (Secció de Quı́mica Orgànica) and Institut de Quı́mica Teòrica i Computacional (IQTCUB) , Universitat de Barcelona , Martí i Franquès 1 , 08028 Barcelona , Spain.

Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcal·mol. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.
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http://dx.doi.org/10.1021/jacs.9b09655DOI Listing
February 2020

Modeling catalytic reaction mechanisms in glycoside hydrolases.

Curr Opin Chem Biol 2019 12 12;53:183-191. Epub 2019 Nov 12.

Departament de Química Inorgànica I Orgànica (secció de Química Orgànica) and Institut de Química Teòrica I Computacional (IQTCUB), Universitat de Barcelona, Martí I Franquès 1 08028 Barcelona, Spain; Institució Catalana de Recerca I Estudis Avançats (ICREA) Passeig Lluís Companys 23, 08010 Barcelona, Spain. Electronic address:

Modeling catalysis in carbohydrate-active enzymes is a daunting challenge because of the high flexibility and diversity of both enzymes and carbohydrates. Glycoside hydrolases (GHs) are an illustrative example, where conformational changes and subtle interactions have been shown to be critical for catalysis. GHs have pivotal roles in industry (e.g. biofuel or detergent production) and biomedicine (e.g. targets for cancer and diabetes), and thus, a huge effort is devoted to unveil their molecular mechanisms. Besides experimental techniques, computational methods have served to provide an in-depth understanding of GH mechanisms, capturing complex reaction coordinates and the conformational itineraries that substrates follow during the whole catalytic pathway, providing a framework that ultimately may assist the engineering of these enzymes and the design of new inhibitors.
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http://dx.doi.org/10.1016/j.cbpa.2019.09.007DOI Listing
December 2019

Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Secretomes Using Activity-Based Probes.

ACS Cent Sci 2019 Jun 24;5(6):1067-1078. Epub 2019 May 24.

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and -β-1,4-xylanases in the secretomes of , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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http://dx.doi.org/10.1021/acscentsci.9b00221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598175PMC
June 2019

Palladium-mediated enzyme activation suggests multiphase initiation of glycogenesis.

Nature 2018 11 24;563(7730):235-240. Epub 2018 Oct 24.

Department of Chemistry, University of Oxford, Oxford, UK.

Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease, and mouse knockout and inherited human mutations of GYG impair glycogen synthesis. GYG acts as a 'seed core' for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195. Precise mechanistic studies have so far been prevented by an inability to access homogeneous glycoforms of this protein, which unusually acts as both catalyst and substrate. Here we show that unprecedented direct access to different, homogeneously glucosylated states of GYG can be accomplished through a palladium-mediated enzyme activation 'shunt' process using on-protein C-C bond formation. Careful mimicry of GYG intermediates recapitulates catalytic activity at distinct stages, which in turn allows discovery of triphasic kinetics and substrate plasticity in GYG's use of sugar substrates. This reveals a tolerant but 'proof-read' mechanism that underlies the precision of this metabolic process. The present demonstration of direct, chemically controlled access to intermediate states of active enzymes suggests that such ligation-dependent activation could be a powerful tool in the study of mechanism.
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http://dx.doi.org/10.1038/s41586-018-0644-7DOI Listing
November 2018

Molecular-Scale Ligand Effects in Small Gold-Thiolate Nanoclusters.

J Am Chem Soc 2018 11 30;140(45):15430-15436. Epub 2018 Oct 30.

Department of Chemistry , Dalhousie University , Halifax , Nova Scotia B3H 4R2 , Canada.

Because of the small size and large surface area of thiolate-protected Au nanoclusters (NCs), the protecting ligands are expected to play a substantial role in modulating the structure and properties, particularly in the solution phase. However, little is known on how thiolate ligands explicitly modulate the structural properties of the NCs at atomic level, even though this information is critical for predicting the performance of Au NCs in application settings including as a catalyst interacting with small molecules and as a sensor interacting with biomolecular systems. Here, we report a combined experimental and theoretical study, using synchrotron X-ray spectroscopy and quantum mechanics/molecular mechanics simulations, that investigates how the protecting ligands impact the structure and properties of small Au(SR) NCs. Two representative ligand types, smaller aliphatic cyclohexanethiolate and larger hydrophilic glutathione, are selected, and their structures are followed experimentally in both solid and solution phases. It was found that cyclohexanethiolate ligands are significantly perturbed by toluene solvent molecules, resulting in structural changes that cause disorder on the surface of Au(SR) NCs. In particular, large surface cavities in the ligand shell are created by interactions between toluene and cyclohexanethiolate. The appearance of these small molecule-accessible sites on the  NC surface demonstrates the ability of Au NCs to act as a catalyst for organic phase reactions. In contrast, glutathione ligands encapsulate the Au NC core via intermolecular interactions, minimizing structural changes caused by interactions with water molecules. The much better protection from glutathione ligands imparts a rigidified surface and ligand structure, making the NCs desirable for biomedical applications due to the high stability and also offering a structural-based explanation for the enhanced photoluminescence often reported for glutathione-protected Au NCs.
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http://dx.doi.org/10.1021/jacs.8b09440DOI Listing
November 2018

Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4.

ACS Cent Sci 2018 Sep 14;4(9):1274-1290. Epub 2018 Sep 14.

BIFI, University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, Zaragoza 50018, Spain.

Mucin-type -glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
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http://dx.doi.org/10.1021/acscentsci.8b00488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161044PMC
September 2018

Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.

ACS Cent Sci 2017 Nov 13;3(11):1168-1173. Epub 2017 Nov 13.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, His) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-His. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from and pantothenate synthetase.
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http://dx.doi.org/10.1021/acscentsci.7b00341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704290PMC
November 2017

The Molecular Mechanism of Substrate Recognition and Catalysis of the Membrane Acyltransferase PatA from Mycobacteria.

ACS Chem Biol 2018 01 11;13(1):131-140. Epub 2017 Dec 11.

Structural Biology Unit, CIC bioGUNE , Bizkaia Technology Park, 48160 Derio, Spain.

Glycolipids play a central role in a variety of important biological processes in all living organisms. PatA is a membrane acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements, and virulence factors of Mycobacterium tuberculosis. PatA catalyzes the transfer of a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to the 2-position of inositol in PIM/PIM. We report here the crystal structure of PatA in the presence of 6-O-palmitoyl-α-d-mannopyranoside, unraveling the acceptor binding mechanism. The acceptor mannose ring localizes in a cavity at the end of a surface-exposed long groove where the active site is located, whereas the palmitate moiety accommodates into a hydrophobic pocket deeply buried in the α/β core of the protein. Both fatty acyl chains of the PIM acceptor are essential for the reaction to take place, highlighting their critical role in the generation of a competent active site. By the use of combined structural and quantum-mechanics/molecular-mechanics (QM/MM) metadynamics, we unravel the catalytic mechanism of PatA at the atomic-electronic level. Our study provides a detailed structural rationale for a stepwise reaction, with the generation of a tetrahedral transition state for the rate-determining step. Finally, the crystal structure of PatA in the presence of β-d-mannopyranose and palmitate suggests an inhibitory mechanism for the enzyme, providing exciting possibilities for inhibitor design and the discovery of chemotherapeutic agents against this major human pathogen.
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http://dx.doi.org/10.1021/acschembio.7b00578DOI Listing
January 2018

1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor.

ACS Cent Sci 2017 Jul 13;3(7):784-793. Epub 2017 Jul 13.

Department of Bio-organic Synthesis and Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6--cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose C chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
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http://dx.doi.org/10.1021/acscentsci.7b00214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532717PMC
July 2017

Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach.

Chembiochem 2017 08 26;18(15):1496-1501. Epub 2017 Jun 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, York, YO10 5DD, UK.

The varied yet family-specific conformational pathways used by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tightly binding and specific enzyme inhibitors. A cardinal example of a GH-family-specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low-nanomolar inhibitor that is selective for GH family 47 inverting α-mannosidases. Here we show, through quantum-mechanical approaches, that kifunensine is restrained to a "ring-flipped" C conformation with another accessible, but higher-energy, region around the B conformation. The conformations of kifunensine in complex with a range of GH47 enzymes-including an atomic-level resolution (1 Å) structure of kifunensine with Caulobacter sp. CkGH47 reported herein and with GH family 38 and 92 α-mannosidases-were mapped onto the kifunensine free-energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymes but cannot mimic any conformational states relevant to the reaction coordinate of mannosidases from other families.
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http://dx.doi.org/10.1002/cbic.201700166DOI Listing
August 2017

Carba-cyclophellitols Are Neutral Retaining-Glucosidase Inhibitors.

J Am Chem Soc 2017 05 5;139(19):6534-6537. Epub 2017 May 5.

Department of Chemistry, University of York , Heslington, York, YO10 5DD, U.K.

The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine-both covalent retaining β-glucosidase inhibitors-we postulated that the corresponding carba "cyclopropyl" analogue would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the H transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the H conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (K = 8.2 nM) of the Thermotoga maritima TmGH1 β-glucosidase. 3-D structural analysis and comparison with unreacted epoxides show that this compound indeed binds in the H conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
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http://dx.doi.org/10.1021/jacs.7b01773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437670PMC
May 2017

The molecular mechanism of the ligand exchange reaction of an antibody against a glutathione-coated gold cluster.

Nanoscale 2017 Mar;9(9):3121-3127

Departament de Química Inorgànica i Orgànica (Secció Química Orgànica) & Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain. and Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys, 23, 08020 Barcelona, Spain.

The labeling of proteins with heavy atom clusters is of paramount importance in biomedical research, but its detailed molecular mechanism remains unknown. Here we uncover it for the particular case of the anti-influenza N9 neuraminidase NC10 antibody against a glutathione-coated gold cluster by means of ab initio QM/MM calculations. We show that the labeling reaction follows an associative double S2-like reaction mechanism, involving a proton transfer, with low activation barriers only if one of the two distinct peptide/peptidic ligands (the one that occupies the side position) is substituted. Positively charged residues in the vicinity of the incoming thiol result in strong interactions between the antibody and the AuMPC, favoring the ligand exchange reaction for suitable protein mutants. These results pave the way for future investigations aimed at engineering biomolecules to increase their reactivity towards a desired gold atom cluster.
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http://dx.doi.org/10.1039/c6nr08498bDOI Listing
March 2017

A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism.

ACS Cent Sci 2016 Dec 8;2(12):896-903. Epub 2016 Nov 8.

York Structural Biology Laboratory, Department of Chemistry, University of York , Heslington, YO10 5DD, U.K.

The enzymatic cleavage of β-1,4-mannans is achieved by -β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 -β-1,4-mannanase from a sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a → → conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.
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http://dx.doi.org/10.1021/acscentsci.6b00232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5200933PMC
December 2016

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase.

J Am Chem Soc 2017 01 17;139(3):1089-1097. Epub 2017 Jan 17.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville 3010, Australia.

Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar "epoxide" mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (K 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.
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http://dx.doi.org/10.1021/jacs.6b10075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5269645PMC
January 2017

Selective Derivatization of N-Terminal Cysteines Using Cyclopentenediones.

Org Lett 2016 10 9;18(19):4836-4839. Epub 2016 Sep 9.

Institució Catalana de Recerca i Estudis Avançats (ICREA) , Passeig Lluis Companys 23, 08010 Barcelona, Spain.

The outcome of the Michael-type reaction between thiols and 2,2-disubstituted cyclopentenediones varies depending on the thiol. Stable compounds with two fused rings were formed upon reaction with 1,2-aminothiols (such as N-terminal cysteines in peptides). Other thiols gave reversibly Michael-type adducts that were in equilibrium with the starting materials. This differential reactivity allows differently placed cysteines to be distinguished and has been exploited to prepare bioconjugates incorporating two or three different moieties.
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http://dx.doi.org/10.1021/acs.orglett.6b02301DOI Listing
October 2016

A Trapped Covalent Intermediate of a Glycoside Hydrolase on the Pathway to Transglycosylation. Insights from Experiments and Quantum Mechanics/Molecular Mechanics Simulations.

J Am Chem Soc 2016 Mar 1;138(10):3325-32. Epub 2016 Mar 1.

Departament de Química Inorgànica i Orgànica and Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona , Martí i Franquès 1, 08028 Barcelona, Spain.

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG(⧧) = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 → [(4)E](⧧) → (1,4)B/(4)E to (4)C1 → [(4)H3](⧧) → (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.
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http://dx.doi.org/10.1021/jacs.5b10092DOI Listing
March 2016

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases.

Chem Sci 2015 Feb 27;6(2):1167-1177. Epub 2014 Oct 27.

Departament de Química Orgànica and Institut de Química Teòrica i Computacional (IQTCUB) , Universitat de Barcelona , Martí i Franquès 1 , 08028 Barcelona , Spain . Email:

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of metadynamics, using the Cremer-Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, S → [B] → S and S → [H] → C, which clearly appear in low energy regions of the FEL. Consistently, S and S are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the E → [S] → B itinerary that has been recently proposed for a family 11 xylanase. Classical and QM/MM molecular dynamics simulations reveal that, in this case, the observed E conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases.
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http://dx.doi.org/10.1039/c4sc02240hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811086PMC
February 2015