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Heparan sulfate analogues act as decoy receptors and efficiently block enterovirus 71 infection Daniel Earley, Benjamin Bailly, Andrea Maggioini, Avinash Kundur, Robin J. Thomson, Chih-Wei CHANG, and Mark von Itzstein ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00070 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Infectious Diseases

Heparan sulfate analogues act as decoy receptors and efficiently block enterovirus 71 infection Daniel F. Earley,‡,1 Benjamin Bailly,‡,1 Andrea Maggioni,1 Avinash R. Kundur,2 Robin J. Thomson,1 Chih-Wei Chang1* and Mark von Itzstein1* Institute for Glycomics, Griffith University, Gold Coast, Queensland, 4222, Australia School of Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD 4222, Australia Contact email: [email protected] 1 2

Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease, for which there is no antiviral therapy. We have developed densely sulfated disaccharide heparan sulfate (HS) analogues that are potent small molecule inhibitors of EV71 infection that bind the viral capsid and act as decoy receptors to block early events of virus replication. The simplified structures, more potent than defined HS disaccharides and with no significant anticoagulant activity, offer promise as anti-EV71 agents. Keywords: enterovirus 71(EV71), Hand foot and mouth disease (HFMD), heparan sulfate, binding inhibitor, HS analogue, sulfation fragments has not, to the best of our knowledge, been reported. Well-designed HS-based disaccharides could potentially match the specific binding parameters of HS required for interaction with EV71, and provide increased antiviral potency, as compared to full-length GAGs. We have therefore explored the potential of disaccharide HS fragments and mimetics to act as inhibitors of EV71 in vitro infection. We synthesized a series of defined, regioselectively sulfated HS disaccharides (Chart 1b) to probe the effect of extent of sulfation and sulfation pattern on interaction with the virus and infection blockade. We then progressively increased the extent of sulfation and demonstrated that highly-sulfated HS disaccharide analogues (Chart 1c) block early stages of virus-cell interaction and are potent inhibitors of infection. The synthesized small molecule inhibitors have significantly improved antiviral activity when compared to large GAG and GAG-like inhibitors such as HS and fondaparinux, respectively, and unlike large sulfated polysaccharides lack significant anticoagulant activity.

Hand, foot, and mouth disease (HFMD) is a highly contagious, generally self-limiting illness commonly observed in children. It is caused by infection with enterovirus A species, that include enterovirus 71 (EV71) and coxsackievirus A16. HFMD is characterized by a papulovesicular rash of the palms and soles, and multiple oral ulcers.1 In rare cases, however, EV71 infection is associated with severe neurological disease and systemic complications that can be fatal.2,3 EV71 has caused sporadic outbreaks of HFMD worldwide that have been increasingly prevalent across the Asia-Pacific region, where it has become a major public health issue.4,5 There are currently no specific antiviral agents available for the treatment of EV71 infection. The increasing incidence of HFMD outbreaks, and the potential for severe complications, is now driving antiviral drug discovery research to combat enterovirus A infection, in particular EV71.6 Among the antiviral strategies explored is the development of compounds that target viral entry into host cells. Several cellular receptors have been identified that aid EV71 attachment to, and infection of, the target host cells of the gastrointestinal and respiratory tracts. These include the ubiquitous human scavenger receptor class B2 (SCARB2), human P-selectin glycoprotein ligand-1 (PSGL-1), annexin II (Anx2), sialylated glycans and sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS).7–11 HS is a large, negatively-charged, linear polysaccharide, covalently bound to a core protein to form an HS proteoglycan (HSPG). HSPGs are major functional constituents of the extracellular matrix and are involved in a range of biological processes.12–14 HS itself is comprized of repeating disaccharide units of -D-glucosamine (GlcN, N) and either -D-glucuronic (GlcA, G) or -L-iduronic acid (IdoA, I), and features high levels of O- and N- sulfation (Chart 1a).15 For EV71 infectivity, Tan et al. reported that N- and O-sulfation on HS is essential, and that the degree of sulfation is functionally important.10 Given the role of HS in the initial stages of EV71 infection, large MW anionic polysaccharides have been used to inhibit in vitro infection.16 However, the use of shorter, defined HS-based

Chart 1. Structures of (a) heparan sulfate (HS) disaccharide sequences,a and representative examples of (b) defined HS disaccharides and (c) densely sulfated HS disaccharide analogues, synthesized to explore inhibition of EV71 infection.

a

HS sequences with commonly observed N- (blue) and O- (red) sulfation sites highlighted.

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benzoyl-3-O-benzyl-1,6-anhydro-idopyranose was used as the glycosyl acceptor, under N-iodosuccinimide (NIS)/trimethylsilyl trifluormethanesulfonate (TMSOTf)20 promoted glycosylation. Utilizing the orthogonally protected disaccharides 1 and 2, a small series of defined HS disaccharide fragments (3–8, Chart 2) was synthesized following procedures previously described in the literature.17,20 Each disaccharide was first capped at the reducing end with the minimal methyl glycoside. Three NG (3– 5) and three NI (6–8) disaccharides were prepared, carrying varying levels of N- and / or O- sulfation, to broadly represent the chemical diversity of HS disaccharides.25 The explicit synthetic procedures for these compounds can be found in the Supporting Information.

RESULTS AND DISCUSSION Synthesis of defined HS disaccharide fragments. Despite the abundance of HS in mammalian biology, pure HS fragments can be challenging to isolate from natural sources.17 As a consequence, chemo-enzymatic or chemical syntheses have been used to produce fragments of defined sequence and sulfation pattern.17–19 Our strategy for the synthesis of defined disaccharide HS fragments for studies with EV71, and for HS mimetics, utilized two orthogonally-protected HS disaccharide precursors, compounds 1 and 2 (Chart 2). These are composed of functionalized monosaccharide subunits prepared from Dglucosamine and D-glucose (for 1) or L-idose (for 2). The target HS disaccharide fragments GlcN--(1,4)-GlcA (NG) and GlcN--(1,4)-IdoA (NI) as well the potential N- and Osulfation sites (represented by blue and red ellipses, respectively) are depicted in Chart 1a. The orthogonal protecting group strategy used for disaccharides 1 and 2, developed with reference to procedures reported by a number of research groups in this area,17,20–24 was designed to allow independent manipulation of key functional groups, leading to greater flexibility in the synthesis of both the HS fragments and HS mimetics. A combination of temporary and more permanent protecting groups was used in particular to facilitate the introduction of the specific O-sulfation patterns of HS (Figure 1).

Chart 2. Overview of the synthetic approach used for the preparation of HS disaccharides 3 to 8 via orthogonally protected precursors.a

Figure 1. Overview of the protecting groups and potential functional group elaboration for the NG disaccharide precursor 1.

A tert-butyldiphenylsilyl (TBDPS) ether was used to protect the C-6 hydroxy group of the GlcN subunit, for future selective installation of a 6-O-sulfate group. Similarly, the orthogonal benzoyl (Bz) group was employed to allow selective installation of a 2-O-sulfate on the GlcA and IdoA residues. The C-2 amine of the GlcN subunit was masked as an azide, firstly to take advantage of the azide's non-participating character in the initial monosaccharide glycosylation (favoring the desired -anomer), and secondly, as upon reduction the resultant amine can be converted selectively to either an acetamido or sulfonatoamino functionality as required. The C-3 hydroxy groups of both the GlcN and GlcA/IdoA precursor subunits, which did not require further modification, were protected with permanent benzyl (Bn) protecting groups. The C-4 hydroxy group of the GlcN subunit was protected as the 2naphthylmethyl ether (Nap). Finally, the C-6 primary hydroxy group of the Glc or Ido subunit was protected by an acetyl (Ac) group, for selective deprotection and subsequent oxidation to the corresponding carboxylic acid of the GlcA or IdoA subunit. An overview of the synthetic approach to the HS disaccharide precursors 1 and 2, and their subsequent conversion to a number of defined HS disaccharides, is shown in Chart 2. For the synthesis of 1, the fully protected 2-azido-2deoxy-glucopyranosyl trichloroacetimidate glycosyl donor (highlighted in yellow) was reacted with the thiotolyl glucoside acceptor, catalyzed by silver trifluoromethanesulfonate (AgOTf).24 For the synthesis of 2, the thiotoyl glycoside of 2azido-2-deoxy-glucose was the glycosyl donor, while 2-O-

a The three key monosaccharide building blocks used as precursors to GlcN (N), GlcA (G) and IdoA (I) subunits are highlighted in yellow, green and cyan respectively.

HS disaccharide fragments inhibit EV71 in vitro infection. The synthesized HS disaccharide fragments 3–8 were screened for their ability to block EV71 in vitro infection of rhabdomyosarcoma (RD) cells. To screen for antiviral activity, test compounds were evaluated at concentrations of 2 mM, and 200 and 20 M. The sulfated glycosaminoglycan HS, which has been identified as an inhibitor of EV71 in vitro infection that blocks virus attachment to cells,26 was used as a benchmark and was found to have an IC50 value of 102.1 ± 12.2 g/mL (Figure 2a), in good agreement with published values.16 Using compounds 3–8, we could compare the antiviral potencies of compounds based on the NG and NI scaffolds, containing various patterns and levels of sulfation. As shown in 2


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ACS Infectious Diseases HS disaccharide fragments bind to the EV71 capsid in a sulfate-dependent manner. To assess the binding interactions of selected HS fragments with EV71 we have used saturation transfer difference (STD) NMR spectroscopy.26,27 This technique has been previously used to study a range of glycan receptor–virus interactions and determine receptor-binding epitopes.28,29 Figure 3 presents the reference 1H and STD-NMR spectra of compound 5 (Figure 3a), compound 8 (Figure 3b) and fondaparinux (Figure 3c), in the presence of purified EV71 particles at pH 7.4, 283 K. Fondaparinux (Figure 3d) is a synthetic, pentasaccharide, low molecular weight heparin analogue that features eight sulfate groups. It is comprised of the sulfated disaccharide sequences of 5 (A+B) and 3'-Osulfated 8 (C+D), terminated by an additional di-sulfated GlcN residue [N(NS,6S)--OMe]. It has previously been reported to be an inhibitor of EV71 in vitro infection.30 In the present study, we determined its IC50 to be 7.5 M (Table 1).

Figure 2b, NG-based compounds 4 [N(NS)G(2S)] and 5 [N(NS,6S)G], which each possess two sulfate groups, displayed a greater antiviral effect than compound 3 [N(NAc,6S)G] which possesses only one sulfate group (29% and 28% infection inhibition at 2 mM for compounds 4 and 5, respectively, and 0% for compound 3). Compounds 4 and 5 each possess both an N- and an O-sulfate group, with compound 4 featuring a 2-Osulfate on the GlcA subunit, while compound 5 presents a 6-Osulfate on the GlcN residue. The different placement of the Osulfate does not appear to influence activity. The inhibitory activity of the NI-based compounds followed a similar pattern to that observed for the NG scaffold, whereby increasing the number of sulfate groups from two in compounds 6 [N(NS)I(2S)] and 7 [N(NS,6S)I] to three in compound 8 [N(NS,6S)I(2S)] significantly improved antiviral potency (21% and 7% infection inhibition at 2 mM for compounds 6 and 7, respectively, and 69% for compound 8). However, unlike the NG-based compounds, a change in the position of O-sulfation from 2-O on IdoA for compound 6, to 6-O on GlcN for compound 7 resulted in a 3-fold loss in inhibitory potency (at 2 mM). This data suggests that the disaccharide composition affects antiviral activity, with NG-based compounds having slightly better activity than NI-based compounds. Together, these results demonstrated that, while not reaching the inhibition levels of the HS polymer (e.g. for 8, 69% inhibition is achieved at 2 mM, or 1395 g/mL; for HS, 50% inhibition is achieved at 102.1 g/mL), there is potential for HS disaccharide fragments to inhibit EV71 in vitro infection, and that increasing the sulfation content may improve their antiviral potency.

Figure 3. 1H and STD-NMR spectra of HS fragments (a) 5 [N(NS,6S)G], (b) 8 [N(NS,6S)I(2S)], and (c) fondaparinux (fpx) [N(NS,6S)GN(NS,3S,6S)I(2S)N(NS,6S)] in the presence of EV71 particles. NMR experiments were carried out in deuterated PBS at pH 7.4 at 283 K, in the presence of 2.5 mM compound. Reference off-resonance proton (1H) and saturation transfer difference (STD) NMR intensities are respectively comparable for all compounds. STD spectra were obtained by subtraction of on-resonance spectra from off-resonance spectra, and peak intensities are representative of the relative binding of ligand protons to the EV71 capsid. *impurity. (d) Structure of fondaparinux. The positions of anomeric protons are labelled from A to E, in red.

Figure 2. Inhibitory activity of HS and selected disaccharide HS fragments against EV71 in vitro infection. (a) Dose-dependent inhibition of HS against EV71 in vitro infection of RD cells. Data points represent the average of triplicate experiments, ± SD (n = 3). (b) Primary screening of HS fragments 3–8 against EV71 in vitro infection of RD cells. Bars represent the average of triplicate measurements, ± SD. 2000 µM corresponds to compound concentrations of 1071, 1191, and 1395 µg/mL for 3, 4–7, and 8 respectively; 200 µM: 107, 119, and 139 µg/mL; 20 µM: 10.7, 11.9, and 13.9 µg/mL. Compound at indicated concentrations was incubated with virus and RD cells on ice for 1 h, after which infection was carried out for 24 h at 35 °C. Infection was measured by in situ ELISA against EV71 VP1.

In the STD spectrum of compound 5 [N(NS,6S)G] (Figure 3a), the signal intensities of the anomeric protons [H-1 (G) and H-1' (N)] are relatively weak, which suggests that they have little interaction with the viral capsid. The signals 3



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Scheme 1. Synthesis of 9 and 13

corresponding to protons H-2' and H-6' on carbons bearing Nand O-sulfate groups respectively are not visible in the STD spectrum, indicating that they are not in close proximity to the viral capsid. The remaining protons however, including those associated with the aglycon methyl group, appear to interact with the viral capsid as indicated by their corresponding STD signals. A comparison of the 1H and STD spectra of compound 8 [N(NS,6S)I(2S)] (Figure 3b) indicates interaction of both anomeric protons [H-1 (I) and H-1' (N)] with the viral capsid. All other carbohydrate residue protons (H-2 to H-5 and H-2' to H-6'), as well as the aglycon methyl group protons have corresponding STD effects. This suggests that the increase in charge density in compound 8 compared to compound 5 leads to an increased binding affinity with the viral capsid. Similarly, for fondaparinux (Figure 3c) all carbohydrate ring protons have corresponding STD signals, including the anomeric protons H1A to H-1D. This indicates a clear interaction of fondaparinux with the viral capsid, most likely due to the high negative charge density provided by its eight sulfate groups. The difference in peak intensity between its 1H and STD spectra is also higher than for compounds 5 and 8. This suggests that although binding is observed for compounds 5 and 8, the binding of fondaparinux occurs with stronger affinity. We hypothesize that the observed high binding affinity (Figure 3) of fondaparinux may be attributed to two structural features: (1) charge density, with fondaparinux having an overall charge density that averages 2 negatively charged groups per residue, which is greater than compound 5 (1.5 negative charges per residue) and equivalent to 8 (2 negative charges per residue); and (2) an increased anchoring footprint over five residues, allowing for better traction on the viral surface compared to compounds 5 and 8. The higher binding affinity of fondaparinux, as indicated by the strong peak intensities of fondaparinux in its STD spectrum, is supported by the relative inhibitory activities of the three compounds examined. Where 5 and 8 inhibit 28% and 69% of in vitro infection at 2 mM, respectively (Figure 2), fondaparinux inhibits 100% of infection at 1 mM (IC50 9.4 M; for doseresponse curve see SI, Figure S1). Synthesis of densely sulfated disaccharide HS mimetics. The results from initial screening of the disaccharide HS fragments against EV71 indicated that increasing the sulfation content on each disaccharide scaffold increased both the antiviral activity (Figure 2) and the EV71 binding affinity (Figure 3), in line with the report of Tan et al.10 It was hypothesized that further increasing the extent of sulfation for both the NG and NI scaffolds could increase inhibitory activity against EV71. Per-O-sulfation of the HS disaccharides meant that both NG and NI scaffolds could accommodate a maximum of five O-sulfate groups for the N-acetyl derivatives. For the NG scaffold, the 6'-O-sulfated N-acetyl-NG disaccharide 3 [N(NHAc,6S)G] was directly subjected to Osulfation using sulfur trioxide trimethylamine complex in anh DMF,31 to afford the per-O-sulfated N(NHAc)G analogue 9 (23%; Scheme 1). For the NI scaffold, synthesis began with the reported idurono-2,6-lactone containing disaccharide 10.20 A three-step deprotection procedure was employed, starting with the removal of the TBDPS ether, followed by sodium hydroperoxide catalysed lactone hydrolysis32 and finally hydrogenolysis, to afford the globally deprotected intermediate 11 (68% over 3 steps). Selective N-acetylation33 of 11 gave compound 12 (99%), which was subsequently per-O-sulfated to afford the penta-O-sulfated N(NHAc)I analogue 13 (54%).

Reagents and conditions: (a) SO3.Me3N, anh DMF, 60 °C, 16 h (9, 23%; 13, 54%); (b) (i) 1 M TBAF in THF solution, 80% aq AcOH, THF, 50 °C, 5 h; (ii) 2 M aq NaOH, 10 M aq H2O2, THF, rt, 2 h; (iii) Pd(OH)2, 20 mM aq NaH2PO4 solution, H2, MeOH, rt, 3 d (68% over 3 steps); (c) Ac2O, Et3N, MeOH, H2O, 5 °C, 16 h (99%).

A second series of densely sulfated disaccharide HS mimetics could be obtained by replacing the uronic acid carboxylate group of the NG and NI scaffolds, with an Osulfated hydroxymethyl group on the related GlcN-Glc and GlcN-Ido scaffolds (Scheme 2). In conjunction with N-sulfation on the GlcN residue, a total of seven sulfate groups on a single disaccharide could be installed, the maximum number possible. The starting point for these syntheses were partially protected intermediates used in the preparation of the natural HS disaccharide units 3-8 (Chart 2). For the GlcN-Glc scaffold the partially protected intermediate 14, and for the GlcN-Ido scaffold the corresponding intermediate 15, were treated with tetrabutylammonium fluoride (TBAF) and 80% aq AcOH in THF at reflux20 to furnish the de-silylated intermediates 16 and 17 respectively (Scheme 2). Subsequent hydrogenolysis gave the globally deprotected derivatives 18 (87% over 2 steps) and 19 (81% over 2 steps), which were then subject to global Osulfation31 to furnish the hexa-O-sulfated disaccharides 20 (GlcN-Glc; 24%) and 21 (GlcN-Ido; 46%). Finally, Nsulfation31 afforded the per-O-sulfated 2'-sulfonatoamino disaccharides HS mimetics 22 (75%) and 23 (33%).

4


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ACS Infectious Diseases

Scheme 2. Synthesis of 21–23

alamarBlue assay (values are mean CC50 ± SD, n = 2) dSI: Selectivity Index, 𝑆𝐼 = 𝐶𝐶50/𝐼𝐶50. nd: not determined. As can be seen from the data in Table 1 for the related NG (9) and NG-like GlcN-Glc (22) scaffolds, the increased charge density and sulfate content of compound 22 significantly improves (3.5-fold) the antiviral activity over compound 9. The per-sulfated HS mimetic 22 has a comparable IC50 value of 7.9 M against EV71 in vitro infection, to that of the pentasaccharide fondaparinux (IC50 9.4 M). For the NI and related NI-like GlcN-Ido scaffolds, there is only a slight improvement in potency as the charge density increases and the number of sulfate groups is increased from five in compound 13 to seven in compound 23 (IC50 values: 16.9 M and 12.1 M, respectively). However, a distinct detrimental effect on inhibitory activity is observed when the GlcN nitrogen is unsubstituted, with compound 21 having ~2-fold weaker activity than N-sulfated analogue 23. This may be due to a decrease in the net negative charge of the molecule, or to some adverse binding interaction of the free amino group of 21. The antiviral potency of per-sulfated GlcN-Glc disaccharide 22 is similar to that of GlcN-Ido analogue 23, although marginally improved (1.5-fold). Compound 22 has an IC50 value of 7.9 ± 3.9 M, or 8.5 ± 4.2 g/mL, and an IC90 value of 43 ± 12 µM, or 46 ± 13 g/mL. In comparison, the IC50 values for HS (Figure 2a) and heparin, both isolated from porcine mucosa, are 102.1 ± 12.2 g/mL and 142.8 ± 42.7 g/mL, respectively. Compound 22 is therefore 12-fold more potent than the natural ligand HS, which demonstrates a higher binding efficiency for the shorter HS disaccharide mimetic. In addition to having low-micromolar potency against EV71 infection, the per-sulfated HS mimetics 22 and 23 have no observable toxicity towards RD cells, even at concentrations as high as 1 mM. The selectivity indices of 22 and 23 were recorded as greater than 127 and 83, respectively, providing excellent in vitro toxicological profiles. To investigate the stability of these polysulfated disaccharides we took a representative compound, compound 22, and stored it in D2O for 96 h at 20 °C. NMR spectra were acquired at 24 h and 96 h (SI, Figure S2), with analysis indicating that the compound was stable over 96 h under these conditions. Synthetic disaccharide HS mimetics lack significant anticoagulant activity. Given the known anticoagulant properties of heparin and HS, the HS mimetic disaccharides 22 and 23 were both evaluated for anticoagulant activity in activated partial thromboplastin time (aPTT) and partial thromboplastin time (PT) coagulation assays. Heparin and low molecular weight heparin (LMWH) were used as positive controls for anticoagulation activity. As shown in Table 2, the concentrations of heparin and LMWH required to double normal human plasma clotting time in the aPTT assay (baseline = 46 ± 3 s) were recorded at 1.7 µg/mL and 2.9 µg/mL, respectively. In the PT assay (baseline = 13 4 ± 0.5 s), the concentrations were 14.5 µg/mL and 27.8 µg/mL for heparin and LMWH respectively. For compounds 22 and 23, the concentrations required to double clotting time in the aPTT assay were 146.1 µg/mL and 151.2 µg/mL respectively; concentrations at least 50-fold greater than seen for heparin/LMWH. At concentrations of 22 and 23 as high as 200 µg/mL, double normal clotting times were not reached in PT assays. These experiments indicate that the HS mimetic disaccharides 22 and 23 do not possess significant anticoagulation properties. This outcome is in line with previous reports showing that highly sulfated disaccharides

Reagents and conditions: (a) 1 M TBAF in THF solution, 80% aq AcOH, THF, reflux, 16 h; (b) Pd(OH)2, 20 mM aq NaH2PO4 solution, H2, MeOH, rt, 3 d (18, 87% over 2 steps from 14; 19, 81% over 2 steps from 15); (c) SO3.Me3N, anh DMF, 60 °C, 16 h (20, 24%; 21, 46%); (d) SO3.Pyr, 0.1 M aq NaOH, Et3N, MeOH, 0 to 5 °C, 18 h (22, 75%; 23, 33%).

Improved anti-EV71 activity of densely sulfated disaccharide HS mimetics. A series of the disaccharide HS mimetics, each containing five to seven sulfate groups, were evaluated in EV71 in vitro infection inhibition assays. The IC50 and IC90 values of the NG-based compounds 9 and 22, and NIbased compounds 13, 21, and 23, with comparison to fondaparinux as a benchmark, are presented in Table 1. Inhibition curves can be found in the SI, Figure S1. As a reference, IC50 values for 3-8 extrapolated from Figure 2b are above 1 mM. Table 1. Antiviral potency (IC50), cytotoxicity (CC50) and selectivity index (SI) of selected disaccharide HS mimetics Compound

Sa

SId

164 ± 29

CC50 (M)c nd

9 (NG)

5

IC50 (M)b 28 ± 14.3

IC90

22 (NG-like)

7

7.9 ± 3.9

43 ± 12

>1000

>127

13 (NI)

5

16.9 ± 6.3

123 ± 68

nd

nd

21 (NI-like)

6

30 ± 7.6

175 ± 70

nd

nd

23 (NI-like)

7

12.1 ± 2.6

129 ± 57

>1000

>83

Fondaparinux

8

9.4 ± 1.8

96 ± 5

nd

nd

(µM)b

nd

aS:

number of sulfate groups. bInhibitory activity against EV71 in vitro infection of RD cells. Cells were infected in the presence of compound for 24 h, after which infection was measured by in situ ELISA (values are mean IC50 or IC90 ± SD, n = 3). cToxicity of compounds to RD cells under the conditions of infection. Cells were incubated with compound for 24 h, after which cell viability was measured by 5



(n = 3). (a, b) Infection was measured by in situ ELISA against EV71 VP1.

such as maltose hexasulfate or sucrose octasulfate are not significantly anticoagulant.34 Most importantly, it demonstrates that compound 22 has an improved pharmacological profile to that of the larger, equipotent pentasaccharide fondaparinux which is a known anticoagulant drug. The anticoagulant property of heparin is due to a pentasaccharide antithrombinbinding sequence from which was derived fondaparinux.35 Therefore, the small size of disaccharide HS mimetics 22 and 23 most likely accounts for their lack of anticoagulant properties.

The data presented in Figure 4a shows that when 22 was present from the time of initial virus adsorption to cells (t–1) or of virus entry (t0), EV71 infection was significantly inhibited (15% and 30% infection, respectively). When added after 1 h of infection (t1) virus replication was only slightly inhibited (75% infection), while no inhibition was observed when 22 was added at later stages of the virus life cycle (t2 to t8). This indicates that compound 22 blocked early events of infection such as virus attachment and subsequent entry to cells, without affecting later-stages such as genome replication or viral protein synthesis. The IC50 value of 22 when present only during virus binding at 4 °C was found to be of the same order of magnitude as its IC50 value when the compound was present throughout infection (Figure 4b; IC50 values: 4.0 ± 0.5 µM and 7.9 ± 3.9 µM, respectively). This demonstrates that the antiEV71 activity of 22 can be attributed to its ability to block early stages of virus–cell interaction, such as virus attachment to cells. Furthermore, adding compound at the post-adsorption stage (1 h after the initial virus infection of cells) resulted in a similar level of inhibition (Figure 4b; IC50 = 3.9 ± 0.6 µM), suggesting that 22 can block subsequent rounds of virus replication. It also supports its potential therapeutic value. We therefore propose that the HS mimetic disaccharides bind to the viral capsid, preventing virus attachment to extracellular GAGs such as HS, and possibly to other receptors of the host cell.

Table 2. Anticoagulant activity of per-sulfated disaccharide HS mimetics. Compound

aPTT

PT

22 (NG-like)

146.1

> 200

23 (NI-like)

151.2

> 200

LMWH

2.9

27.8

Heparin

1.7

14.5

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Values represent compound concentrations in µg/mL required to double normal plasma clotting time. Clotting time baselines measured in aPTT and PT assays: 46 ± 3 s and 13 4 ± 0.5 s, respectively. Disaccharide HS mimetics block early events of viruscell interaction. The STD-NMR experiments presented in Figure 3 demonstrated that the disaccharide HS fragments bind to the EV71 capsid. In light of this result, we proposed that the disaccharide HS mimetics would interfere with infection by blocking virus attachment to cells. We therefore conducted time of compound addition experiments using hepta-sulfated GlcNGlc disaccharide 22, the most potent compound identified in this study. A fixed concentration of 22 (200 µM, approximately 2 × IC99) was added to infected cells at different time points during a single round of virus replication (6 to 8 h).

CONCLUSION Despite the increased understanding of how GAGs such as HS engage with EV71, this avenue has not been extensively investigated for drug discovery purposes. Here, we have exploited the antiviral properties of the naturally occurring GAGs HS and heparin,16 by synthesizing a range of disaccharide HS fragments and mimetics. In screening a small library of defined HS fragments, we discovered that both sulfation content and disaccharide composition are important for inhibition of EV71 infection. This outcome was confirmed by STD-NMR spectroscopy experiments. Capitalizing on this finding, we synthesized a second series of more densely sulfated compounds that included non-natural sulfation patterns. These HS mimetic disaccharides were found to have low µM potency against EV71 in vitro infection, with the most potent being a hepta-sulfated GlcN-Glc disaccharide (22; IC50 7.9 M), which, importantly, lacked significant anticoagulant properties. We demonstrated that 22 blocked EV71 infection by inhibiting early stages of virus-cell interaction, suggesting that the compound binds to the viral capsid and interferes with the virus binding to cellular receptors. This is the first study to report the synthesis and evaluation of disaccharide HS fragments and mimetics as inhibitors of EV71 in vitro infection. The structure-activity relationship studies that we have conducted have led to the synthesis of densely sulfated disaccharides of high inhibitory activity. Impressively these inhibitors deliver either improved or equipotent levels of inhibition to GAGs such as HS and GAG analogue counterparts such as fondaparinux, respectively. The relative structural simplicity of the densely sulfated GlcN-Glc or GlcN-Ido disaccharides eliminates the multiple protecting group manipulations that are otherwise required for the synthesis of natural GAG structures with defined sulfation patterns.

Figure 4. Inhibitory activity of compound 22 at different stages of EV71 in vitro infection. (a) Time of addition-dependent inhibitory activity of compound 22 during a single round of EV71 in vitro replication. RD cells were infected synchronously for 1 h at 4 °C (t–1). At t0 cells were washed, inocula replaced with fresh medium and cells incubated for a single round of virus replication (8 h). Cells were treated with 200 M of compound at the indicated time points. Infection in all wells was stopped at t8. Bars represent the average of duplicate measurements, ± SD. (b) Dose-dependent inhibition of 22 against EV71 in vitro infection. Compound at indicated concentrations was incubated with virus and RD cells for 1 h at 4°C, after which cells were transferred to 35 °C (all stages of infection) or cells were washed and inocula replaced with fresh medium alone (binding 4 °C). Post-adsorption: cells were incubated with virus for 1 h at 35 °C, washed and then treated with compound dilution. Infections were carried out for 24 h at 35 °C. Data points represent the average of triplicate experiments ± SD

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ACS Infectious Diseases analysis using the software GraphPad Prism (GraphPad Software, La Jolla, California, USA). A detailed description of the procedure can be found in SI. General Procedure for O-sulfation. To a solution of selectively de-protected disaccharide in anh DMF (0.03 M), sulfur trioxide trimethylamine complex (3 to 12 equiv per hydroxyl group) was added. The reaction mixture was stirred under an inert atmosphere at 60 ºC for 16 h (monitored by 1H NMR), quenched with Et3N, filtered and concentrated in vacuo (water bath temp of 32 ºC). The crude residue was purified by size-exclusion chromatography [Sephadex LH20 (MeOH) or superfine Sephadex G25 (H2O)] to afford the O-sulfated product (e.g. compound 20; yields and spectral characterization are given in SI).31 General Procedure for N-sulfation. To a 0 ºC solution of amino-disaccharide in MeOH (0.03 M) was added sulfur trioxide pyridine complex (SO3Pyr; 5 equiv), 0.1 M aq NaOH (5 equiv) and Et3N (50 mL per mmol). Additional aliquots of SO3Pyr were added after 1 h and 2 h of continuous stirring. The reaction mixture was then stirred at 5 ºC until completion (monitored by 1H NMR) and concentrated in vacuo (water bath temp of 32 ºC). The crude residue was taken up in a small volume of H2O, filtered (cotton wool) and subject to ionexchange chromatography (Dowex 50WX8 Na+ resin) followed by size-exclusion chromatography (superfine Sephadex G25). The collected fractions were lyophilized to afford the target disaccharide as the sodium salt (e.g. compound 22; yields and spectral characterization are given in SI).31

Our proof of concept study suggests that disaccharide HS mimetics can serve as new scaffolds for the development of EV71 entry inhibitors, and that further modification and optimisation could lead to inhibitors with improved potency. While it has been shown that not all strains of EV71 bind HS with the same affinity,10,36 our synthetic inhibitors are much smaller than natural HS, are structurally defined, and demonstrate an apparently stronger interaction with the virus capsid than HS. While it can be expected that they will block infection of multiple EV71 strains, the spectrum of their activity will need to be further evaluated. Since a number of viral pathogens, such as human respiratory syncytial virus, human parainfluenza virus type-3 and human immunodeficiency virus, also recognise HS to initiate infection,37–39 disaccharide HS mimetics such as those described here could also provide a starting point for the development of antiviral compounds targeting other HS-recognizing viruses.

EXPERIMENTAL SECTION General. A full description of materials and methods is provided in the Supporting Information (SI). Purities of synthetic intermediates after chromatographic purification were judged to be >90% by analysis of 1H and 13C NMR spectra (SI). Purity of final compounds were ≥95% (NMR analysis), after size-exclusion chromatography (Sephadex G25). Methods for in vitro infection inhibition (measured by in situ ELISA)40 and saturation transfer difference (STD) NMR41 assays were adapted from previous reports. Cells and viruses. Human rhabdomyoscarcoma cells (RD, ATCC ref. CCL 136) were maintained in DMEM supplemented with 5% FBS, 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Human enterovirus 71 (EV71) H strain (ATCC ref. VR-1432) was passaged in RD cells in the conditions described above, with 2% FBS (infection medium). In vitro infection inhibition assays. Compounds were tested in technical triplicates, in 96-well plates. Confluent RD cells were infected with 660 ffu/well of EV71 in the presence of compound dilutions for 1 h at 4 °C, with gentle shaking every 15 min. The plates were subsequently transferred to 35 °C and left to incubate for 24 h. Infection was then stopped and measured by in situ ELISA. In situ ELISA. Briefly, infected cells in 96-well plates were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. Cells were then permeabilized and endogenous peroxidases inhibited by addition of IGEPAL and H2O2, respectively, at a final concentration of 1% for 20 min at 37 °C. For the immuno-labeling of EV71 VP1 antigens, cells were incubated with a 1:2000 dilution of primary anti-EV71 VP1 antibody (clone MAB979, Merck Millipore, Darmstadt, Germany) followed by a 1:6000 dilution of secondary goat antiMouse IgG(H+L)-HRP-conjugated antibody (BioRad, Hercules, CA, USA). EV71 VP1 was detected using 50 µL of OptEIA TMB substrate (BD Biosciences, San Jose, CA, USA) and the reaction was stopped by addition of 25 µL of 0.6 M H2SO4 in each well. The absorbances at 450 nM were read using an X-Mark Microplate Absorbance Spectrophotometer (BioRad). The background absorbance in negative control wells (no infection) was subtracted from all other wells and the resulting absorbances were normalised to the absorbance of positive control wells (infection without compound). Infection was expressed as percent of control. Fifty and ninety percent inhibitory concentrations of compounds (IC50 and IC90, respectively) were determined from non-linear regression

ASSOCIATED CONTENT Supporting Information The Supporting Information includes experimental procedures and spectral data for all synthesized compounds, detailed protocols for virus stock production and titration, in situ ELISA, time of compound addition, cell viability and STD-NMR assays, and virus purification for STD-NMR. It also includes dose-response curves corresponding to the data reported in Table 1. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Authors Phone: +61 7 5552 7025. Email: [email protected]

Author Contributions ‡ D.F.E.

and B.B. contributed equally to this study. D.F.E. and C.W.C. synthesized and characterized the HS disaccharides and mimetics. B.B. performed the biological assays and analyzed STDNMR data. A.M. performed the STD-NMR experiments. B.B and A.R.K. performed the coagulation assays. C.-W.C. and R.J.T. oversaw the chemistry and B.B. oversaw the biology aspects of the study. M.v.I. conceived and oversaw the project. D.F.E. and B.B. prepared the manuscript, which was reviewed by all authors. C.W.C. and M.v.I. are co-senior authors.

Funding Sources M.v.I. gratefully acknowledges the National Health and Medical Research Council for financial support (Grant No.1071659). B.B. acknowledges support from a Griffith University New Researcher Grant. D.F.E. thanks the Australian Government for the award of a Postgraduate Research Scholarship.

ACKNOWLEDGEMENTS 7



The authors would like to thank Dr. Wendy Loa-Kum-Cheung at the Griffith Institute for Drug Discovery, Griffith University for performing high accuracy mass spectrometry measurements of the synthesized compounds.

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ABBREVIATIONS

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EV71, enterovirus 71; GAGs; glycosaminoglycans; GlcA (G), D-glucuronic acid; GlcN (N), -D-glucosamine; HFMD, hand, foot,

and mouth disease; HS, heparan sulfate; IC50, 50% inhibitory concentration; IdoA (I), -L-iduronic acid; NG, GlcN--(1-4)GlcA-; NI, GlcN--(1-4)-IdoA-; RD, human rhabdomyoscarcoma cells; STD (NMR), saturation transfer difference (NMR spectroscopy); VP, viral protein.

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