Antiviral Agents from Multivalent Presentation of Sialyl

Mar 7, 2016 - Bioinspired brush polymers containing α-2,6-linked sialic acids at the side chain termini were synthesized by protection-group-free, ...
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Antiviral Agents from Multivalent Presentation of Sialyl Oligosaccharides on Brush Polymers Shengchang Tang,† Wendy B. Puryear,‡,§ Brian M. Seifried,† Xuehui Dong,† Jonathan A. Runstadler,‡,§ Katharina Ribbeck,‡ and Bradley D. Olsen*,† †

Department of Chemical Engineering, ‡Department of Biological Engineering, and §Division of Comparative Medicine, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: Bioinspired brush polymers containing α-2,6-linked sialic acids at the side chain termini were synthesized by protection-group-free, ring-opening metathesis polymerization. Polymers showed strain-selective antiviral activity through multivalent presentation of the sialosides. The multivalent effect was further controlled by independently varying the degree of polymerization, the number density of sialic acids, and the length of side chains in the brush polymers. Optimizing the three-dimensional sialoside spacing for better binding to hemagglutinin trimers was of critical importance to enhance the multivalent effect and the antiviral activity determined by hemagglutination inhibition assays and in vitro infection assays. By taking advantage of their structural similarities with native mucins, these brush polymers can be used as model systems to dissect the intricate design principles in natural mucins.

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Mucin’s protein backbone contains alternating hydrophilic and hydrophobic domains that are chemically chain-extended through disulfide linkages to reach megadalton molar mass.15 Oligosaccharides, often terminated with SAs, are covalently attached to the protein backbone through O-linkages to serine and threonine residues.15 While the structural details of mucins have been gradually revealed, it remains unclear why nature synthesizes mucins with a brush architecture and if this brush structure has advantages over other polymeric counterparts. Therefore, the use of synthetic systems to elucidate the structure−property relationships in multivalent binding of brush polymers has the potential to provide insight into the natural materials and yield synthetic systems with improved antiviral activities. Recently, ring-opening metathesis polymerization (ROMP) has emerged as a robust strategy for synthesizing brush polymers with precisely defined molecular structure and diverse functionalities.16−21 Macromonomers (MMs) with strained norbornene end groups can be polymerized with the use of the third generation Grubbs catalyst (H2IMes) (pyr)2(Cl)2Ru CHPh, which enables synthesis of brush polymers with molar mass up to several millions.16 In addition, such “graftingthrough” strategies have demonstrated high tolerance for unprotected functional groups, which allows facile incorporation of a myriad of biological functional motifs, such as smallmolecule drugs,17 peptides,22 glycans,23 and nucleotides18 at

nfluenza viruses are a major source of infectious disease, causing approximately 250000−500000 deaths annually.1 As a key step in the process of infection, influenza viruses utilize hemagglutinin (HA) trimers embedded in the viral surface membrane to bind to various forms of sialic acid (SA) receptors on the surface of host cells.2 This initial recognition event can be disrupted by the presence of other end-sialyl oligosaccharides from various biopolymers. Mucin, a key proteoglycan component of the mucosal layer covering epithelial cells in many parts of human body, contains a significant fraction of SAs.2 The mucosal layer has been proven to serve as an effective antiviral barrier, preventing virus invasion through physical sequestration. After binding, viruses are removed by the clearance of mucus every several hours.2,3 The HA-SA interaction also mediates infection by some bacterial pathogens such as Pseudomonas aeruginosa.4 The multivalent presentation of SAs to bind viruses present in mucin has inspired several designs of antiviral polymers via covalently attaching SA residues onto a polymeric scaffold. Since the earliest work from Whitesides and co-workers,5 a large variety of polymeric scaffolds have been explored, including linear6 and dendritic7 polymers, liposomes,8 and polymersomes.9 Through multivalent presentation of SAs, binding between SA clusters and HAs is enhanced by up to several orders of magnitude, thermodynamically and kinetically.10,11 The multivalent effect has also found numerous applications in other areas such as inflammatory regulation,12 signal transduction,13 and cancer therapies.14 Despite the successes of multivalent binding, the reason why nature uses mucins with brush polymer structures as its own multivalent antiviral scaffolds has not yet been deciphered. © XXXX American Chemical Society

Received: December 14, 2015 Accepted: March 4, 2016

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ACS Macro Letters

new physical chemistry due specifically to the brush architecture. The entire synthesis method does not involve any protection-group chemistry yet maintains the controllability of ROMP, enabling efficient, scalable preparation of oligosaccharide-containing brush polymers. The structurally dependent antiviral activities are evaluated by hemagglutination inhibition (HAI) assays and in vitro infection assays, and the results are compared to commercially available bovine submaxillary mucins. Comparison of the changes in molecular architecture illustrates specific advantages of brush polymers that nature exploits for multivalent presentation. MMs with terminal SAs were synthesized by clicking sialyllactose onto the chain end of a norbornenyl macromonomer (Scheme 1b). First, α-norbornenyl, ω-acetylene poly(ethylene glycol) (PEG) was prepared from commercially available amine-PEG-hydroxyl (Mn = 3500 g/mol, Đ = 1.03). The amine group was first modified by reacting with norbornenyl glycine N-hydroxysuccinamide (NHS) ester to install a polymerizable handle, yielding MM 1. The remaining hydroxyl chain end was subsequently esterified with 4pentynoic acid to incorporate a clickable alkyne moiety. Separately, azido oligosaccharides were prepared using the method previously described.29−31 Specifically, the anomeric hydroxyl group of 6′-sialyllatose was activated by 2-chloro-1,3dimethylimidazolinium (DMC) and diisopropylethylamine (DIPEA), which was then converted to azide by nucleophilic substitution. Residual sodium azide was completely removed by dialysis, as confirmed by FT-IR (Figure S1). 1H NMR revealed that 90% of the products were the β-anomer, that is, with the azide group appearing in the equatorial position (Figure S2), consistent with a previous study.30 Finally, MM 4 was prepared using an efficient copper catalyzed “click” chemistry mediated by tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA). Pure product was obtained after purification by HPLC to remove unreacted materials and copper complexes. The structure of MM 4 was confirmed by 1H NMR (Figure S3) and MALDI-TOF MS (Figure S4). Brush homopolymers from MM 4 were then prepared through ROMP, which yielded the desired molecular structure. Here, N,N-dimethylformamide (DMF) was chosen as the polymerization solvent to ensure good solubility of MMs and resulting brush polymers. Although the presence of dense unprotected functional groups on the trisaccharide moieties might render ROMP challenging, the polymerization proceeded to high conversion (>95% for all cases, Table 1). This presents a significant synthetic advancement compared to current strategies for preparing ROMP polymers with pendant carbohydrate side groups that rely on postmodification32 or protection chemistries.33,34 More importantly, ln([M]0/[M]) was found to be linearly proportional to reaction time t, demonstrating characteristics of living polymerizations (Figure S5 for P50). Polymers with varying backbone DPs (25, 50, and 100) were successfully synthesized by changing the ratio of MM to catalyst [MM]/[C] (Table 1). A small high-molecularweight shoulder appeared in the gel permeation chromatography (GPC; Figure S6), most likely due to relatively poor kinetic control when the polymerization mixtures gelled at high conversions (especially for higher targeted DPs), a phenomenon previously observed in ROMP of carboxylic acid containing MMs in DMF.22 It could also originate from the existence of trace impurities of telechelic dinorbornenyl MMs.35 Nonetheless, all polymers have dispersities Đ less than 1.3 (Table 1).

very high density (nearly 100% stoichiometry with the monomer unit). Herein, we employ ROMP to synthesize brush polymers with SA moieties decorating at the side chain termini to maximize their accessibility24 (Scheme 1a). We systematically Scheme 1. Synthetic Detailsa

a

(a) Preparation of bioinspired brush polymers containing terminally attached sialic acids via ring-opening metathesis polymerization. (b) Synthetic routes to norbornenyl (in blue) macromonomers containing sialic acids (in green). (c) Chemical structure of macromonomers with a triethylene glycol backbone.

vary three aspects of the molecular structure, namely, the degree of polymerization (DP) of the backbone, the number of oligosaccharides per polymer, and the side chain length. While the effects of these molecular parameters on the antiviral activities have been investigated extensively in other multivalent scaffolds,25−28 brush polymers access regimes of molecular design space that are different than previously studied systems due to their longer side chains, extremely high ligand density, and relatively rigid backbone; these structural features necessitate systematic experimental investigation to reveal 414

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ACS Macro Letters Table 1. Characterization of Brush Polymers Containing SAs entry

polymer

MM type

[MM]0 (M)

[MM]/[C]

Mn,theo.b (kDa)

Mn,GPCc (kDa)

ĐM d

conv.e (%)

1 2 3 4 5 6 7 8

P25 P50 P100 C25a C50a O25 O50 O100

4 4 4 1+4 1+4 5 5 5

0.05 0.025 0.01 0.01 0.01 0.05 0.05 0.05

25 50 100 100 100 25 50 100

107.9 218.0 422.8 352.6 396.0 26.4 52.8 105.6

107.8 237.2 414.7 468.9 683.7 28.2 57.8 118.6

1.16 1.26 1.26 1.28 1.18 1.03 1.06 1.18

97 98 95 89 96 > 99 > 99 > 99

The numbers 25 and 50 indicate the percentage of MM 4 in the copolymerization reaction mixture. bMn,theo = Mn,MM × [MM]/[C] × conv. cMn,GPC was determined using specific refractive index increment (dn/dc) 0.076 mL/g for polymeric MM 4 and 0.12 mL/g for oligomeric MM 5. dDispersity was determined by GPC MALLS analysis. eConversion was calculated based on the peak areas of MMs and brush polymers from the differential refractive index measurement in GPC. a

To vary the number density of SA in the brush polymers, MMs 1 and 4 were copolymerized at different ratios (75:25 and 50:50), while the targeted backbone DP was kept at 100. Given the length of the PEG chain separating the reactive norbornene from the other end group, it is reasoned that these two MMs were statistically incorporated. While copolymerization kinetics were not rigorously measured in this work, this hypothesis has been previously proven.36 MM 5 (Scheme 1c) containing a shorter triethylene glycol spacer between SA and the norbornenyl end group was prepared for studying the effect of side chain length (see synthetic details in the Supporting Information). Interestingly, polymers of these short MMs remained soluble throughout the polymerization period, and no shoulder was observed in the GPC traces (Figure S7). This evidence supports the previous hypothesis that gelation interferes with polymerization control for high-molar-mass brush polymers. The antiviral activities of brush polymers are highly dependent on their molecular structure, as revealed from hemagglutination inhibition (HAI) assays using human influenza strain A/WSN/1933(H1N1) as a model virus (Figure 1). While no hemagglutination inhibition was observed for the entire concentration range of monovalent SAs, brush polymers exhibited inhibition at varying concentrations, enabling the formation of buttons as virions bound to polymers rather than erythrocytes. The inhibition constant Ki5 was used to compare the activities among different polymers. Ki was assigned as the SA concentration in the mixture at the highest dilution for which inhibition was still achieved, as indicated by an arrow in Figure 1. While Ki for monovalent SA was reported to be of the order 1 mM5, Kis for potent brush polymers were generally 2−4 orders of magnitude smaller (i.e., in the μM range), confirming that antiviral activities were enhanced through the multivalent effect. By comparing homopolymers P25, P50, and P100, it is clear that a larger backbone DP strengthens the HA−SA interactions, so the smallest Ki (0.24 μM) was observed for P100. When the total concentration of SAs was kept constant, but the number of SAs per polymer was decreased by a factor of 2 or 4, hemagglutination inhibition was achieved only at a higher SA concentration (C50 vs P50) or was not detectable under the experimental condition (C25 vs P25). Furthermore, the length of the brush side chains was also important to polymers’ inhibition performance. Polymers with shorter side chains (O25, O50, and O100) exhibited Kis that were 2 orders of magnitude larger than their counterparts with longer arms. Optimization of the multivalent effect hinges on matching the spatial distribution of HAs on viruses with the three-

Figure 1. Antiviral activities of polymers assessed by the hemagglutination inhibition (HAI) assay using A/WSN/1933(H1N1) (abbreviated as WSN) and A/PR/8/34(H1N1) (abbreviated as PR8, the last column and the last row in the figure) as model influenza strains. Wells in the same column contained the same concentration of SAs. The inhibition constant Ki was determined from the lowest SA concentration that maintains complete hemagglutination inhibition. Ki larger than 62.5 μM could not be revealed in the experiment setup and was thus denoted as n.d. (not detectable).

dimensional presentation of SAs on brush polymers. Although thoroughly rationalizing the multivalent effect remains challenging, three heuristic design rules are informed from this study. First, the distance between SA residues should be smaller than the intra-HA spacing (ca. 4.5 nm37) to display higher local concentrations of SAs so that various binding modes10 such as chelation and statistical effect are possible. This requirement was fulfilled for all polymers. Second, polymers with a contour length larger than the inter-HA spacing (ca. 14 nm37) are able to bridge multiple HA trimers, thus, not only providing an additional benefit of steric shielding, but also increasing the possibility of statistical rebinding to HA trimers to enhance multivalency. By assuming that brush polymers adopt a flexible cylindrical conformation and that the backbone length per monomer unit is 0.18 nm,38 this criterion was only met for polymers with a DP of 100. Third, longer arms are expected to reduce entropic penalty from the conformational changes of polymers when binding to HAs, which might also enhance multivalency. Taking all these three aspects together, it is not surprising that P100, which had the 415

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Figure 2. (a) Schematic illustration of the in vitro infection assay. Objects are not drawn to scale. (b) Fluorescence images (from left to right) showing noninfected MDCK cells and cells infected by viruses, under the protection layer of polymer solutions of P100 and mucins, and in the absence of polymers. Scale bar: 10 μm. (c) Bar charts showing the percentages of infected cells counted by flow cytometry. SA concentration were fixed at 0.5 mM for all polymer solutions. Experiments were performed in triplicate and results were reported as mean ± standard deviation. Significance level (p value) was calculated from the student t test, *p < 0.05, **p < 0.01, ***p < 0.001. (d) Concentration dependence of polymer P100 on infection suppression. Error bars represent standard deviation from triplicated measurements. The solid line shows a linear fit in the concentration range from 0.005−1 mg/mL. The dashed line shows the infection rate without addition of polymers. The arrow points to concentration at which comparative studies in (c) were done.

sugar moiety.31,40 For example, human influenza viruses preferentially bind to α-2,6-linked SAs whereas avian influenza viruses preferentially bind to α-2,3-linked SAs.40 More interestingly, even both A/WSN/1933(H1N1) and A/PR/8/ 34(H1N1) are lab adapted human influenza strains, the latter primarily binds to α-2,3-linked SAs. When the PR8 strain was used in the HAI assay, no hemagglutination inhibition was observed for all synthetic brush polymers (see the last column in Figure 1), which clearly indicates the polymers’ binding specificity to selective virus strains depending on their HA structure. On the contrary, BSG mucin showed recognition and inhibition toward both virus strains (see the last two rows in Figure 1) because it has both types of SA linkages. It is therefore envisioned that the SA-containing brush polymers with well-defined structure have a potential to distinguish and even subtype various virus strains, and that mixing of different sialoside isomers may provide broad spectrum antiviral capability. The structure-dependent antiviral effect was also confirmed by in vitro infection assays. Brush polymers were first added on top of modified Madin-Darby canine kidney cells (MDCKSIAT1-CMV-PB1), forming a pseudo-protection barrier. Engineered viruses that carry a reporter gene of green fluorescence protein (GFP) were then added to infect the

largest backbone DP, the highest density of SAs and the longest side chains, showed the best inhibition activity. More importantly, all of these design rules might be met by native mucins, which have a high SA density (in the glycosylation region ca. 1/3 of the amino acids, serines and threonines, are O-glycosylated with oligosaccharides terminated with SA39), a moderate arm length (5−15 monomers per oligosaccharide side chain15) and a very large total molecule size (on the order of 100 nm15), covering all relevant length scales to enhance multivalent effect. Successful optimization of the multivalent effect in synthetic brush polymers also leads to better inhibition performance compared to biopolymers from which the design inspiration was drawn. Commercial mucins from bovine submaxillary glands (BSG) were chosen for comparison due to their relative high SA content among available products. The SA content in BSG mucins was experimentally determined to be about 10 wt % using an acidic ninhydrin assay (see Supporting Information for details), which was comparable to those in synthetic brush polymers. Under the same HAI assay condition, mucins showed a Ki that was equivalent to that of P50, but 8× larger than that of P100 (Figure 1). The HA trimers on the virion surface not only recognize the terminal SA residues but also distinguish the linkage to the next 416

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ACS Macro Letters cells at 37 °C for 2 h. Subsequently, both polymers and viruses were carefully washed away, and infected cells were incubated overnight to allow GFP expression (Figure 2a,b). The fraction of infected cells was determined by flow cytometry. This assay simulates the actual infection process3 and thus provides a more biologically- relevant context to interrogate the structure− property relationship of mucin-mimetic brush polymers. Assay conditions were selected so that in the absence of any polymeric barriers, approximately 90% of cells were infected. In contrast, the percentages of infected cells dropped significantly with the addition of SA brush polymers. Consistent with the results from the HAI assays, polymers with a higher backbone DP exhibited lower infection rates (see both the P and O series of brush polymers in Figure 2c). Polymers with longer side chains also appreciably suppressed infection. When compared to BSG mucins, polymer P100 more effectively protected cells from human influenza (p < 0.001). P25 and C50 were statistically indistinguishable from BSG mucins, showing that there is significant process latitude in engineering brush polymers that are at least effective as natural ones. The in vitro infection assays also suggested that the viscosity of brush polymer solutions contribute to suppressing infection. While pronounced differences in Ki were observed in the HAI assays by comparing polymer pairs C50−P50 and C25−P25, respectively (Figure 1), the difference in the in vitro infection rate was statistically insignificant (C50 vs P50) or the infection rate of the copolymer was even unexpectedly lower (C25 vs P25; Figure 2c). These seemingly contradicting results are reconciled by considering the effect of solution viscosity. To keep the total SA concentration constant for all experiments, the polymer concentrations for C25 and C50 were higher than P25 and P50. Diffusive motion of viruses inside the polymer protection layer is affected by the HA-SA affinity interaction (roughly the inverse of Ki) and the solution viscosity. Although the binding interactions were weaker for the two copolymers, an increase in their solution viscosities (Figure S8) suppresses virus diffusion and hence reduces the infection rate. The viscosity effect should also contribute, to some extent, to decreasing the infection rates for polymers with longer side chains (Figure S8). However, it is important to emphasize that the multivalent effect is still the primary factor causing the difference in antiviral activities between P100 and mucin because these two polymers had similar viscosities that were comparable to pure PBS buffer (Figure S8). The most effective concentration range of P100 was then identified from studies of the concentration dependence on the infection rate (Figure 2d). When polymer concentrations were below 1 mg/mL, the percentages of the infected cells showed a linear dependence on the polymer concentration (note that the concentration axis is presented on a logarithmic scale). Further increases in polymer concentration resulted in small decreases in the infection rate that ultimately plateaued at about 3%. In summary, SA-functionalized brush polymers can be synthesized via ROMP in a protection-group-free manner. The synthetic strategy presents an advanced and efficient route to obtain structurally tunable, high-molecular-weight brush polymers with a high density of multivalent ligand. The multivalent inhibition of influenza viruses is probed by the HAI assays and the in vitro infection assays. Polymers with a higher backbone DP, a higher density of SA moieties and longer side chains exhibit stronger antiviral activities. In addition to the thermodynamic aspect, the viscosity of the polymer solution also plays an important role in decreasing the infection rate.

Taking the synthetic brush polymers as a model system for native mucins, these findings suggest a plausible explanation to the question why nature uses brush polymers as the defense scaffold for native mucins: densely grafted side chains are used for multivalent presentation of ligands (thermodynamics), spanning the relevant biological length scales for optimizing ligand−receptor interactions; the long backbone provides a sufficient viscosity (dynamics), especially when the soluble domains are joined with hydrophobic domains forming a multiblock structure and when the polymers are at much higher concentrations under physiological conditions. Because the structure of ROMP polymers can be predictable and various functionalities can be precisely installed, the brush architecture should find opportunities in engineering a wide range of multivalent ligands.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00917. Synthetic procedures and characterization of MMs, and Figures S1−S16 (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Defense Threat Reduction Agency under the Contract HDTRA1-13-1-0038 to S.T., B.M.S., X.D., K.R., and B.D.O., and by the National Institute of Allergy and Infectious Diseases under Contract HHSN272201400008C to W.B.P. and J.A.R. The authors would like to thank Dr. Jesse Bloom (Fred Hutchinson Cancer Research Center) for providing the MDCK-SIAT1-CMV-PB1 cell line and the WSN-PB1flank-eGFP virus strain, Chris Bandoro for assistance with the infection assay, Prof. Jeremiah Johnson for the use of HPLC, and Jenny Liu for training, Prof. Krystyn Van Vliet for the use of microscope, and Prof. Karen Gleason for the use of FT-IR. The authors are also grateful for discussions with Prof. Matthew Gibson (University of Warwick) and Dr. Thomas Crouzier.

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