Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GIcNAc-Man(5)GlcNAc(2) to produce GlcNAcMan(3) GlcNAc(2), the precursor for all complex N-glycans,... Show moreGolgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GIcNAc-Man(5)GlcNAc(2) to produce GlcNAcMan(3) GlcNAc(2), the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce alpha-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of alpha-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors. Show less
Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GIcNAc-Man(5)GlcNAc(2) to produce GlcNAcMan(3) GlcNAc(2), the precursor for all complex N-glycans,... Show moreGolgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GIcNAc-Man(5)GlcNAc(2) to produce GlcNAcMan(3) GlcNAc(2), the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce alpha-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of alpha-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors. Show less
Madern, J.M.; Kim, R.Q.; Misra, M.; Dikic, I.; Zhang, Y.; Ovaa, H.; ... ; Noort, G.J.V. van 2020
Stable NAD(+)analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD(+)-consuming enzymes. To investigate the... Show moreStable NAD(+)analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD(+)-consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD(+)analogues, namely carbanicotinamide adenosine dinucleotide (c-NAD(+)), thionicotinamide adenosine dinucleotide (S-NAD(+)) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD(+)mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD(+)analogues in a scalable manner. We then compared the three NAD(+)mimics side-by-side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S-NAD(+)and BAD with IC(50)values of 28 and 39 mu M, respectively. Show less
Dendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against... Show moreDendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against invading pathogens. PRR ligands are frequently conjugated to tumor-associated antigens in a vaccination strategy to enhance the immune response toward such antigens. One of these PPRs, DC-SIGN, a member of the C-type lectin receptor (CLR) family, has been extensively targeted with Lewis structures and mannose glycans, often presented in multivalent fashion. We synthesized a library of well-defined mannosides (mono-, di-, and tri-mannosides), based on known “high mannose” structures, that we presented in a systematically increasing number of copies (n = 1, 2, 3, or 6), allowing us to simultaneously study the effect of mannoside configuration and multivalency on DC-SIGN binding via Surface Plasmon Resonance (SPR) and flow cytometry. Hexavalent presentation of the clusters showed the highest binding affinity, with the hexa-α1,2-di-mannoside being the most potent ligand. The four highest binding hexavalent mannoside structures were conjugated to a model melanoma gp100-peptide antigen and further equipped with a Toll-like receptor 7 (TLR7)-agonist as adjuvant for DC maturation, creating a trifunctional vaccine conjugate. Interestingly, DC-SIGN affinity of the mannoside clusters did not directly correlate with antigen presentation enhancing properties and the α1,2-di-mannoside cluster with the highest binding affinity in our library even hampered T cell activation. Overall, this systematic study has demonstrated that multivalent glycan presentation can improve DC-SIGN binding but enhanced binding cannot be directly translated into enhanced antigen presentation and the sole assessment of binding affinity is thus insufficient to determine further functional biological activity. Furthermore, we show that well-defined antigen conjugates combining two different PRR ligands can be generated in a modular fashion to increase the effectiveness of vaccine constructs. Show less
Fabry disease is an inherited lysosomal storage disorder that is characterized by a deficiency in lysosomal α-D-galactosidase activity. One current therapeutic strategy involves enzyme replacement... Show moreFabry disease is an inherited lysosomal storage disorder that is characterized by a deficiency in lysosomal α-D-galactosidase activity. One current therapeutic strategy involves enzyme replacement therapy, in which patients are treated with a recombinant enzyme. Co-treatment with enzyme active-site stabilizers is advocated to increase treatment efficacy, a strategy that requires effective and selective enzyme stabilizers. Here, we describe the design and development of an α-D-gal-cyclophellitol cyclosulfamidate as a new class of neutral, conformationally constrained competitive glycosidase inhibitors that act by mimicry of the Michaelis complex conformation. We found that D-galactose-configured α-cyclosulfamidate 4 effectively stabilizes recombinant human α-D-galactosidase (agalsidase beta, Fabrazyme®) both in vitro and in cellulo. Show less
Hendrikse, S.I.S.; Su, L.; Hogervorst, T.P.; Lafleur, R.P.M.; Lou, X.; Marel, G.A. van der; ... ; Meijer, E.W. 2019
Polysaccharides present in the glycocalyx and extracellular matrix are highly important for a multitude of functions. Oligo- and polysaccharides-based biomaterials are being developed to mimic the... Show morePolysaccharides present in the glycocalyx and extracellular matrix are highly important for a multitude of functions. Oligo- and polysaccharides-based biomaterials are being developed to mimic the glycocalyx, but the spatial functionalization of these polysaccharides represents a major challenge. In this paper, a series of benzene-1,3,5-tricarboxamide (BTA) based supramolecular monomers is designed and synthesized with mono- (BTA-β-d-glucose; BTA-Glc and BTA-α-d-mannose; BTA-Man) or disaccharides (BTA-β-d-cellobiose; BTA-Cel) at their periphery or a monosaccharide (BTA-OEG4-α-d-mannose; BTA-OEG4-Man) at the end of a tetraethylene glycol linker. These glycosylated BTAs have been used to generate supramolecular assemblies and it is shown that the nature of the carbohydrate appendage is crucial for the supramolecular (co)polymerization behavior. BTA-Glc and BTA-Man are shown to assemble into micrometers long 1D (bundled) fibers with opposite helicities, whereas BTA-Cel and BTA-OEG4-Man formed small spherical micelles. The latter two monomers are used in a copolymerization approach with BTA-Glc, BTA-Man, or ethylene glycol BTA (BTA-OEG4) to give 1D fibers with BTA-Cel or BTA-OEG4-Man incorporated. Consequently, the carbohydrate appendage influences both the assembly behavior and the internal order. Using this approach it is possible to create 1D-fibers with adjustable saccharide densities exhibiting tailored dynamic exchange profiles. Furthermore, hydrogels with tunable mechanical properties can be achieved, opening up possibilities for the development of multicomponent functional biomaterials. Show less
During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is... Show moreDuring infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multi-functional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh’s highly electronegative binding cleft versus the neutral surface present in Sph3h. Our insight into PelAh’s structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here. Show less
Pre‐activation based glycosylations have become a very powerful tool in the assembly of oligosaccharides and the use of nucleophilic additives allows for the in situ generation of reactive... Show morePre‐activation based glycosylations have become a very powerful tool in the assembly of oligosaccharides and the use of nucleophilic additives allows for the in situ generation of reactive intermediates with tailored reactivity. We here use a glycosylation strategy that is based on the use of per‐benzylated imidate building blocks for the fully stereoselective construction of a spacer equipped Aspergillus fumigatus α‐1,3‐octaglucan. We have used the trimethylsilyl iodide (TMSI)‐triphenylphosphine oxide (Ph3P=O) for the stereoselective installation of an azidopropanol spacer and triflic acid (TfOH)‐dimethyl formamide (DMF) enabled glycosylations for the coupling reactions with the secondary glucosyl C‐3‐alcohols. An operationally simple in situ activation coupling procedure is introduced and used for the final glycosylation events towards the octasaccharide. Show less
Madern, J.M.; Hansen, T.; Rijssel, E.R. van; Kistemaker, H.A.V.; Vorm, S. van der; Overkleeft, H.S.; ... ; Codee, J.D.C. 2019
Thiosugars, sugars that have their endocyclic oxygen substituted for a sulfur atom, have been used as stable bioisosteres of naturally occurring glycans because the thiosugar glycosydic linkage is... Show moreThiosugars, sugars that have their endocyclic oxygen substituted for a sulfur atom, have been used as stable bioisosteres of naturally occurring glycans because the thiosugar glycosydic linkage is supposed to be stabilized toward chemical and enzymatic hydrolysis. We have performed an in-depth investigation into the stability and reactivity of furanosyl thiacarbenium ions, by assessing all four diastereoisomeric thiofuranosides experimentally and computationally. We show that all furanosyl thiacarbenium ions react in a 1,2-cis-selective manner with triethylsilane, reminiscent of their oxo counterparts. The computed conformational space occupied by the thiacarbenium ions is strikingly similar to that of the corresponding furanosyl oxycarbenium ions, indicating that the stereoelectronic substituent effects governing the stability of furanosyl oxocarbenium ions and thiacarbenium ions are very similar. While the thio-ribo-furanose appears to be less reactive than its oxo counterpart, the thio-ara-, lyxo-, and xylo-furanosides appear to be more reactive than their oxygen equivalents. These differences are accounted for using the conformational preference of the donors and the carbocation intermediates. The lower reactivity of the thio-ribo furanosides in (Lewis) acid-mediated reactions and the similarity of the thia- and oxocarbenium ions make thio-ribofuranosides excellent stabilized analogues of the naturally occurring ribo-furanose sugars. Show less
With the picolinyl (Pic) group as a C‐1 located directing group and N3 as versatile precursor for C5‐NH2, a novel 1‐Pic‐5‐N3 thiosialyl donor was designed and synthesized, based on which a new... Show moreWith the picolinyl (Pic) group as a C‐1 located directing group and N3 as versatile precursor for C5‐NH2, a novel 1‐Pic‐5‐N3 thiosialyl donor was designed and synthesized, based on which a new sialylation protocol was established. In comparison to conventional sialylation methods, the new protocol exhibited obvious advantages, including excellent α‐stereoselectivity in the absence of a solvent effect, broad substrate scope encompassing the challenging sialyl 8‐ and 9‐hydroxy groups of sialic acid acceptors, flexibility in sialoside derivative synthesis, high temperature tolerance and easy scalability. In particular, the applicability to the synthesis of complex and bioactive N‐glycan antennae when combined with the MPEP glycosylation protocol via the “latent‐active” strategy has been shown. Mechanistically, the excellent α‐stereoselectivity of the novel sialylation protocol could be attributed to the dramatic electron‐withdrawing effect of the protonated Pic groups, which was supported by control reactions and DFT calculations. Show less
With the picolinyl (Pic) group as a C‐1 located directing group and N3 as versatile precursor for C5‐NH2, a novel 1‐Pic‐5‐N3 thiosialyl donor was designed and synthesized, based on which a new... Show moreWith the picolinyl (Pic) group as a C‐1 located directing group and N3 as versatile precursor for C5‐NH2, a novel 1‐Pic‐5‐N3 thiosialyl donor was designed and synthesized, based on which a new sialylation protocol was established. In comparison to conventional sialylation methods, the new protocol exhibited obvious advantages, including excellent α‐stereoselectivity in the absence of a solvent effect, broad substrate scope encompassing the challenging sialyl 8‐ and 9‐hydroxy groups of sialic acid acceptors, flexibility in sialoside derivative synthesis, high temperature tolerance and easy scalability. In particular, the applicability to the synthesis of complex and bioactive N‐glycan antennae when combined with the MPEP glycosylation protocol via the “latent‐active” strategy has been shown. Mechanistically, the excellent α‐stereoselectivity of the novel sialylation protocol could be attributed to the dramatic electron‐withdrawing effect of the protonated Pic groups, which was supported by control reactions and DFT calculations. Show less
Gerlach, D.; Guo, Y.; De Castro, C.; Kim, S.H.; Schlatterer, K.; Xu, F.F.; ... ; Peschel, A. 2018
Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are... Show moreMethicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5–40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences. Show less
Rooden, E.J. van; Kreekel, R.; Hansen, T.; Janssen, A.P.A.; Esbroeck, A.C.M. van; Dulk, H. den; ... ; Stelt, M. van der 2018
Diacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired.... Show moreDiacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired. Here, we report the design, synthesis and application of a triazole urea probe for DAGL equipped with a norbornene as a biorthogonal handle. The activity and selectivity of the probe was assessed with activity-based protein profiling. This probe was potent against endogenous DAGLα (IC50 = 5 nM) and it was successfully applied as a two-step activity-based probe for labeling of DAGLα using an inverse electron-demand Diels–Alder ligation in living cells. Show less
Vorm, S. van der; Hengst, J.M.A. van; Bakker, M.H.E.; Overkleeft, H.S.; Marel, G.A. van der; Codee, J.D.C. 2018
Enhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine... Show moreEnhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine moieties from a clinical perspective, as they can harbor more than one immunogenic epitope enabling treatment of a broader target population. In addition, longer peptides are not extracellularly loaded on MHC class I; rather, they require intracellular processing and will thereby be presented to T cells mainly by professional APCs, thereby avoiding the risk of tolerance induction. The drawback of peptide vaccines regardless of peptide length is that naked peptides are not actively targeted to and taken up by APCs, and the standard nonconjugated adjuvant-peptide mixtures do not ensure cotargeting of the two to the same APC. We have identified a tetanus toxin–derived B cell epitope that can mediate the formation of immune complexes in the presence of circulating Abs. In this study, we show that these immune complexes improve both Ag uptake by APCs (blood monocytes and CD1c+ dendritic cells) and consequently improve CD8+ T cell recall responses in a human ex vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on Abs and the complement component C1q. We envision that this strategy can be used to facilitate active uptake of Ags into APCs to improve T cell responses against pathogens or cancer. Show less
Es, D. van der; Berni, F.; Hogendorf, W.F.J.; Meeuwenoord, N.; Laverde, D.; Diepen, A. van; ... ; Codee, J.D.C. 2018
