Pentavalent Sialic Acid Conjugates Block Coxsackievirus A24 Variant and Human Adenovirus Type 37-Viruses That Cause Highly Contagious Eye Infections

Article abstract of DOI:10.1021/acschembio.0c00446

Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens.

Full text of DOI:10.1021/acschembio.0c00446

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Article
Pentavalent sialic acid conjugates block coxsackievirus
A24 variant and human adenovirus type 37 –
viruses that cause highly contagious eye infections
Emil Johansson, Rémi Caraballo, Nitesh Mistry, Georg Zocher, Weixing
Qian, C. David Andersson, Daniel L. Hurdiss, Naresh Chandra, Rebecca
Thompson, Lars Frängsmyr, Thilo Stehle, Niklas Arnberg, and Mikael Elofsson
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.0c00446 • Publication Date (Web): 26 Aug 2020
Downloaded from pubs.acs.org on August 26, 2020
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Pentavalent sialic acid conjugates block coxsackievirus A24 variant and
human adenovirus type 37 – viruses that cause highly contagious eye
infections
Emil Johansson,a,b Rémi Caraballo,a,b Nitesh Mistry,c Georg Zocher,d Weixing Qian,a,b,e C. David
Andersson,a,b Daniel L. Hurdiss,f Naresh Chandra,c Rebecca Thompson,g Lars Frängsmyr,c Thilo
Stehle,d,h Niklas Arnbergc and Mikael Elofsson*a,b
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Affiliations
aDepartment of Chemistry, Umeå University, SE90187 Umeå, Sweden.
bUmeå Centre for Microbial Research, Umeå University, SE90187 Umeå, Sweden.
cDepartment of Clinical Microbiology, Section of Virology Umeå University, SE90185 Umeå, Sweden.
dInterfaculty Institute of Biochemistry, University of Tübingen, 72076 Tübingen, Germany.
eChemical Biology Consortium Sweden, Umeå University, SE90185 Umeå, Sweden.
fVirology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine,
Utrecht University, 3584CL Utrecht, The Netherlands.
gAstbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds,
Leeds LS2 9JT, United Kingdom.
hVanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
*Professor Mikael Elofsson
Address: Department of Chemistry, Umeå University, SE90187 Umeå, Sweden
E-mail: mikael.elofsson@umu.se
ORCID
Mikael Elofsson: orcid.org/0000-0002-3219-4669
Table of contents graphic
ABSTRACT
Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative
agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and
epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available
for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize
sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have
shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of
cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their
inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells.
The pentavalent conjugates are the first reported inhibitors of CVA24v infection, and proved efficient in
blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for
the development of general inhibitors of these highly contagious ocular pathogens.
INTRODUCTION
Every year millions of individuals around the world are affected by acute conjunctivitis.1,2 The disease
can be caused by allergens, chemicals, or underlying systemic disease; however, the majority of cases
are caused by viruses and bacteria.1-3 Viral conjunctivitis is estimated to cause up to 80% of all cases
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of acute conjunctivitis.1 Acute hemorrhagic conjunctivitis (AHC) and epidemic keratoconjunctivitis (EKC)
are severe and highly contagious forms of viral conjunctivitis.4,5 AHC is characterized by a sudden onset
of ocular pain, foreign body sensation, excessive lacrimation, keratitis, and conjunctivitis.6 Many of these
symptoms are also shared with EKC and it is difficult to differentiate these diseases in a clinical setting.7
The infections are generally self-limited, resolving in one to two weeks, but can become potentially life-
threatening in immunocompromised individuals.5,8,9 AHC spreads rapidly within communities, affecting
up to 50% of the population. As a result, outbreaks can exhaust healthcare resources of affected regions
and severely disrupt the economy.5,10 To date, neither antiviral agents nor vaccines are available for
treating or preventing AHC or EKC.
AHC is predominantly caused by two members of the Picornaviridae family, Coxsackievirus A24
variant (CVA24v) and enterovirus 70 (EV70).11 CVA24v has been reported as the principal etiological
agent responsible for >10 million cases of AHC worldwide during the last decades, including two
pandemics and numerous recurring outbreaks.10,12 To facilitate attachment and subsequent entry,
members of the Picornaviridae engage a range of different cellular receptors including coxsackie and
adenovirus receptor (CAR), decay accelerating factor (DAF, CD55), low density lipoprotein receptor
(LDL-R), human P-selectin glycoprotein ligand-1 (PSGL-1), heparan sulfate, integrins, intercellular
adhesion molecule-1 (ICAM-1), and glycan-containing receptors terminating in 5-N-acetyl-neuraminic
acid (sialic acid).13-17 EV70 is reported to bind DAF and glycans that terminate in α2,3-linked sialic
acid,13,16 while CVA24v binds to α2,3- and α2,6-linked sialic acids via pentameric binding sites.14,15,18 In
addition, engagement of the ICAM-1 receptor is essential for coxsackievirus A24 infection, including
nonvariant and variant strains of the virus. However, the virulence and pandemic nature of the variant,
AHC-causing, strain seems to be linked to an enhanced ability to bind sialic acid.19 This adaptation has
been suggested to enable efficient attachment of CVA24v to corneal and conjunctival cells,15 and
thereby enhance the transmission of viral progeny.19
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EKC is contracted by millions of individuals worldwide every year.20 The disease is caused by
members of the Adenoviridae family (HAdV-8, -19, -37, -53, -56, and -64), with HAdV-37 serving as a
leading causative agent.21 EKC-causing human adenoviruses are reported to engage CD46 or CAR,
and α2,3-linked sialic acids to facilitate attachment.22,23 More recently, it was established that HAdV-37
binds to glycoproteins containing the GD1a glycan motif via its homotrimeric fiber knobs proteins.21
Sialic acid is a highly polar and negatively charged nine-carbon monosaccharide. It is abundantly
expressed on the surface of all cell types and serves important roles in mediating or modulating a variety
of physiological and pathophysiological processes.24 For a number of diseases, it has been proposed
that sialic acid conjugates can be used for therapeutic intervention. However, in general terms
development of carbohydrate-based therapeutics is challenging due to poor pharmacological properties
imparted by their high polarity, metabolic vulnerability, and low affinity protein-carbohydrate interactions.
In addition, preparation of carbohydrates can be both cumbersome and expensive.25 In the case of
CVA24v and HAdV-37 many of these downsides can potentially be circumvented by design of
multivalent ligands26 intended for topical administration e.g. eye drops, creams, or ointments. Design of
multivalent carbohydrate ligands offers an excellent strategy to achieve high avidity binding to lectins26-
28 via the “cluster glycoside effect”27 or proximity/statistical effects28. The cluster effect is caused by the
binding of several monosaccharide ligands to several binding sites on the lectin, while the
proximity/statistical effect is caused by a slower off-rate of a multivalent ligand in comparison to a
monovalent ligand based on reasoning of proximity.28 Pivotal work with Shiga and cholera toxins
demonstrated that multivalent carbohydrate ligands tailored to lectin valency and topology produce
,
dramatic enhancements in avidity.29 30 The bacterial AB5 Shiga toxin and radially symmetric multivalent
glycoconjugates were further used to develop and validate a quantitative model to analyze
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thermodynamic parameters of multivalent interactions.
To date, the only substance reported to inhibit attachment of CVA24v to cells is a 13-valent sialic
acid human serum albumin conjugate.15 For HAdV-37, we and others have shown that trivalent and
multivalent sialic acid conjugates are potent inhibitors HAdV-37 attachment and infection.32-34 In this
study, we developed novel pentavalent sialic acid conjugates with potential to act as general inhibitors
of both AHC and EKC. Such inhibitors could be applied via topical administration and used for treatment
and prophylactics to alleviate experienced symptoms, and to limit transmission of these highly
contagious infections.
RESULTS AND DISCUSSION
Design of pentavalent sialic acid conjugate
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We previously reported that trivalent sialic acid conjugates efficiently inhibit HAdV-37 infection of human
corneal epithelial (HCE) cells.32,33 These inhibitors were designed to chelate the sialic acid binding
sites,33 which are arranged in a symmetric trimeric fashion in the knob domain of the HAdV-37 12 fibers
protruding from the virus particle. We reasoned that a similar strategy could be applied to CVA24v. In
contrast to HAdV-37, CVA24v has five sialic acid binding sites organized over each of the 12 relatively
flat surfaces of the virus particle.18 Thus, we hypothesized that the sialic acid binding sites of CVA24v
could effectively be chelated by pentavalent sialic acid conjugates (Figure 1A). Such pentavalent
inhibitors would also have potential to act on HAdV-37 by providing a high local concentration of sialic
acid upon binding to the virion by engaging one to three of the sialic acid binding sites of the fiber knob
protein with the non-engaged sialic acids in close proximity (Figure 1B).
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Figure 1. Design of pentavalent sialic acid conjugates
A) Schematic representation of a CVA24v particle, and close-up view of one of the pentameric sialic
acid binding sites with inhibitor modelled. Inhibitor (purple) with highlight of sialic acid (yellow). B)
Schematic representation of a HAdV-37 particle, and zoom-in of one of the fiber knob proteins that
contain trimeric sialic acid binding sites with inhibitor modelled. Inhibitor (purple) with two sialic acids in
solution (grey) and three sialic acids binding (yellow). C) Schematic design of the pentavalent sialic acid
conjugates. The sialic acid residues are connected to a common core fragment via spacers of sufficient
length for chelation of the pentameric binding sites of CVA24v.
Perpropargylated D-glucose was selected as the core fragment (Figure 1C),35 as it allows rapid access
to pentavalent sialic acid conjugates via “click” chemistry. Further, glucosides can be accessed in either
α- or β-configuration opening an interesting opportunity to study potential differences in binding mode
and potency between the two anomers. Two spacer fragments (a polyethylene glycol (PEG) fragment
and an amide containing alkyl fragment) were set to contain an azide function in one end and a hydroxyl
group in the other. The length of these spacers can be tuned to study the effect of spacer length on
potency.32,33,36 The design was validated by molecular modelling of pentavalent conjugates 25–28
(Scheme 1) to CVA24v (Figure S1). The resulting complexes were subjected to conformational searches
followed by energy minimizations of the spacers. The final conformations were evaluated, and no steric
clashes between the conjugates and CVA24v were evident and the carbon-carbon bond lengths and
angles showed no strain and were in agreement with benchmark calculations (Table S1). Geometries
for the α- and β-configuration of the core were similar with slightly less strain on the latter.
Synthesis of 1st set of pentavalent conjugates
Synthesis of the pentavalent sialic acid conjugates 25–28 was achieved in four steps from the sialic acid
donors 137 and 237 (Scheme 1). The core fragment 19 (α-core) was synthesized by a two-step procedure
involving microwave-accelerated Fischer glycosylation of D-glucose using propargyl alcohol,38 followed
by O-alkylation35 with an overall yield of 23%. The spacer fragment 3 was synthesized in 95% yield by
substituting the corresponding alkylchloride with an azide using NaN3. Spacer fragment 4 was isolated
in 62% yield by coupling 5-amino-1-pentanol with 3-bromopropionyl chloride, followed by azide
formation (SI). Donor 1 was sialidated using spacer fragment 3 and 4, respectively, under promotion of
silver trifluoromethanesulfonate and IBr39 affording the corresponding azido acetyl protected sialosides.
Subsequent O-deacetylation afforded the α-anomerically pure azido sialosides 11 and 12, respectively,
in 28% and 13% yield over two steps. Compounds 11 and 12 were then “clicked” to the
pentapropargylated glucose cores 19 and 2035 affording the corresponding pentavalent sialic acid
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methyl esters 21–24 in 41–73% yields. Subsequent saponification provided the corresponding
pentavalent sialic acid conjugates 25–28 in 86% to quantitative yields after neutralization.
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Scheme 1. Synthesis of the 1st and 2nd set of pentavalent sialic acid conjugates 25–28 Reagents
and conditions: a) i: Molecular sieves (3Å), 3–9, CH3CN/CH2Cl2 (2:1), rt, 30 min, ii: Silver
trifluoromethanesulfonate, IBr, –74 °C, 5.5 h. b) Sodium methoxide, methanol, rt, 3 h, ii: H+ ion exchange
.
resin. c) CuSO4 5H2O, sodium ascorbate, tetrahydrofuran/H2O (1:1), 19 or 20, 50 °C, 5 h ➝ rt, 18 h. d)
i: LiOH, methanol, rt, 24 h, ii: H+ ion exchange resin.
Pentavalent sialic acid conjugates inhibit CVA24v attachment to HCE cells
To evaluate the biological effect of the pentavalent conjugates binding inhibition assays were
performed.15 Compared with untreated virions, the attachment of 35S-labeled CVA24v whole virus
particles to HCE cells was significantly attenuated when pre-incubated with 25–28 (Figure 2A). All
inhibitors were significantly more potent than sialic acid (IC50 >> 2 mM) and disialyllacto-N-tetraose (50%
reduction in binding at 2 mM).18 Inhibitors 27 and 28 both containing an amide spacer were equally
potent (IC50 159 µM and 163 µM, respectively), while the PEG based inhibitors 25 and 26 showed lower
potency (IC50 423 µM and 670 µM, respectively). Thus, the inhibitors containing an amide spacer were
3- to 4-fold more potent than inhibitors containing a PEG spacer. All inhibitors showed similar absolute
potency to the previously reported 13-valent sialic acid conjugate.15 However, in terms of relative ligand
efficiency, the ratio of the affinity of a ligand divided by the number of heavy atoms in the molecule, 25–
28 were significantly more efficient at inhibiting CVA24v attachment.
Pentavalent sialic acid conjugates inhibit CVA24v infection
To further evaluate the biological effect of the pentavalent conjugates, infection assays were
performed.32 All four pentavalent conjugates 25–28 inhibited CVA24v virus particles from infecting HCE
cells (Figure 2B), thus confirming the trends from the binding assays. Compounds 27–28 were 5- to 11-
fold more potent than 25–26. All inhibitors were significantly more efficient than sialic acid
monosaccharide that showed no apparent effect on infection at 4 °C (Figure 2B).
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Increasing the temperature to physiological conditions (37 °C) did not significantly alter the effect
of the inhibitors on the infection (Figure S2), thus indicating the inhibitors are efficient despite the
decreased utilization of sialic acid at increasing temperature by CVA24v.19
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Figure 2. Effect of pentavalent sialic acid conjugates on CVA24v binding and infection of HCE
cells and binding of 28 to CVA24v penton
A) Virion binding in presence of inhibitors at different concentrations at 4 °C. B) Infection at different
concentrations of inhibitors at 4 °C. Error bars shown as standard error of the mean (SEM). Data are
presented as % of control that is the value obtained in the absence of inhibitor. All experiments were
performed in duplicates for and a minimum of two times. C) Structure of 28-CVA24v complex. Observed
electron density shown in cage, remaining parts of 28 have been modelled for clarity. B) Interactions of
sialic acid moiety of 28 to amino acids in ligand binding site of CVA24v.
X-Ray Crystallography supports binding of the pentavalent sialic acid conjugates to CVA24v
Next, we investigated the binding modes of 25–28 by X-ray crystallography. Structures of all four (25–
28) inhibitor-CVA24v complexes were established with resolutions up to 1.8 Å. All structures showed
clear electron density for the sialic acid in all of the five binding sites, in addition to a couple of the closest
spacer atoms. Remaining parts of the inhibitors were not detected, likely due to high flexibility and
adaptation of several conformations; these have been modelled into the 28-CVA24v complex (Figure
2C). The binding of 25–28 is very similar to the binding observed for other glycan-CVA24v structures
with most of the contacts retained.18 The key contributors to inhibitor binding are Ser147, Tyr250, and
Tyr145 that directly interact with the sialic acid units of the inhibitors. Ser147 interacts with the
carboxylate group of sialic acid by donating hydrogen bonds from its side chain hydroxyl group and its
main chain amide proton. The side chain of Tyr250 donates a hydrogen bond and a water-mediated
hydrogen bond to the C5-acetamido carbonyl oxygen and the C4 hydroxyl group of sialic acid,
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respectively. The main chain amido carbonyl of Tyr145 accepts a hydrogen bond from the C5
acetamido-NH group of sialic acid. Moreover, a water-mediated hydrogen bond is contributed by the
carbonyl oxygen of Pro252. To stabilize the conformation of Tyr145, an extensive π-π stacking network
involving Tyr145, Arg254, and Phe95 appears important for enabling contacts to the sialic acid unit.
Hydrophobic contacts are also contributed by Tyr145, Ala146, and Pro252 non-polar regions of the
binding site. The C9 hydroxyl of the sialic acid entity of 28 engage in intramolecular hydrogen bonding
to the carboxylate group causing the glycerol chain to adopt a conformation that previously were
observed only as a minor conformation for other glycan containing structures 4Q4X and 4Q4Y (Figure
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2D). On the basis of the structural data, an explanation to this peculiar interaction could not be
determined. The absence of electron density for the core and spacer fragments of 25–28 are in line with
previous observations with trivalent sialic acid conjugates containing long and flexible spacer
fragments.32 In that case, successive shortening of the spacers, resulted in decreased flexibility and
increased potency, eventually resulting in detection of density for entire molecules including spacers
and core.33
Design and synthesis of the 2nd set of pentavalent sialic acid conjugates
The second set of compounds focused on studying the effect of spacer length on potency. Due to their
higher potency, inhibitors containing an amide appeared more attractive for probing of a tailor-made
chelation of the sialic acid binding sites. Thus, compounds 39–42 containing successively shorter
spacers and 43–46 containing longer spacers were designed (Figure 3A). Placement of the amide was
probed by moving it closer to the sialic acid residues in compounds 39–42.
Compounds 40 and 46, containing the shortest and longest amide containing spacers,
respectively, were subjected for further evaluation using computational methods (Figure S3 and Table
S1). In an analogous manner to the 1st set, modelling of 40 and 46 to CVA24v including conformational
searches and energy minimizations, indicated that the spacers were of sufficient length to not cause
strain on the spacers; nor were steric clashes with the amino acid backbone of the virion evident (Table
S1 and Figure S3, respectively). Thus, the designed compounds 39–46 could be used to probe for the
optimal length required for a tailor-made chelation of the binding sites.
To challenge the spacer length 47 and 48 were designed (Figure 3A). Modelling of 48 to CVA24v
showed severe strain, manifested by a large average C-C-C bond angle of 123.3° (Figure S3 and Table
S1). In addition, steric clashes occurred between the aliphatic spacers of 48 and amino acid residues of
the virion thus indicating that although possible, simultaneous binding of all five sialic acid residues to
the pentameric binding sites of CVA24v is associated with significant energy costs. Therefore, the
potency of 48 should be significantly decreased as the length of its spacers are unlikely to be of optimal
length for chelating the sialic acid binding sites. In contrast, 47 is unlikely to act as an optimal chelator
due to its extended length, and its potency should therefore be reduced as binding is likely associated
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with large entropic costs due to significant loss of translational and rotational degrees of freedom.
Synthesis of pentavalent conjugates 39–48 was achieved in analogous manner to the synthesis
of 25–28, with minor modifications. The use of donor 2 in combination with decreased concentration of
spacer fragment 4 proved beneficial in the synthesis of azido sialoside 12, improving the yield to 35%
over two steps. Therefore, donor 2 was used in the synthesis of azido sialosides 13–18.
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Figure 3. Effect of spacer length on CVA24v and HAd-V37 binding to HCE cells, and effect of 28
on thermal stability of CVA24v. A) CVA24v binding at 4 °C in presence of inhibitors at different
concentrations. For clarity, only representative examples from the series of spacer optimized
compounds are illustrated in the diagram. Binding inhibition data for all pentavalent sialic acid conjugates
25–28 and 39–48 are summarized in the table and their corresponding curves can be found in Fig. S3.
Error bars shown as SEM. Data are presented as % of control that is the value obtained in the absence
of inhibitor. All experiments were performed in duplicates for and a minimum of two times. B) Effect of
28 on thermal stability of CVA24v. Fluorescence curves of CVA24v with (solid black line) and without
(solid grey line) 28. The dashed lines correspond to fluorescence of 28 in absence of CVA24v (black),
and fluorescence of denatured CVA24v (grey line). Table below graph shows summary of CVA24v Tm
and Tm shifts (∆Tm) as measured for protein (measured by SYPRO orange) and RNA with and without
treatment of 100 µM sialic acid, pleconaril, or 28. All graphs are presented in the SI. C) HAd-V37 binding
at 4 °C in presence or absence of inhibitors at different concentrations. Only representative examples
from the complete series of spacer optimized pentavalent conjugates were tested, in addition to trivalent
sialic acid conjugate ME046233 used as a control. Error bars in A and C are shown as SEM. Data are
presented as % of control that is the value obtained in the absence of inhibitor. All experiments were
performed in duplicates for and a minimum of two times.
Spacer length of the conjugates affects the inhibitory potency against CVA24v
The inhibitory effect of the 2nd set of compounds was evaluated in cell binding assays. The most potent
compound 44 (IC50 157 µM) was measured to be 1.6-fold more potent than reference compound 28
(IC50 248 µM) (Figure 3A). As expected, 47 (IC50 1.16 mM) and 48 (IC50 1.12 mM) with very long or short
spacers were the least efficient of the series proving 5.4- and 4.6-fold less potent than 28 respectively
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(Figure 3A). Compounds 39–42, containing shorter spacers, were 1.8- to 2.1-fold less potent than 28,
while compounds 43–46, containing longer spacers, were equally or 1.4- to 1.6-fold more potent than
28 (Figure 3A, and Figure S4 for dose-response curves). Thus, increased spacer length resulted in
slightly improved potency. Correlation between anomeric configuration of the core and potency could
however not be demonstrated. This can potentially be attributed to the absence of direct contacts
between the protein backbone of the virion and the spacers and core. Further, the compounds are able
to bind with either the α- or the β-face of the core projecting toward the virion thus rendering potential
effects of core configuration to a minimum. However, it is possible that the anomeric configuration might
play a critical role in combination with a certain linker type and length. On the other hand, a weak
correlation between spacer length and potency was observed as compounds with longer spacers were
3- to 4-fold more potent than compounds with shorter spacers. However, greatly increasing the length
of the spacer was not beneficial, reflecting the greater entropic costs of binding of a more flexible ligand.
Spacer type did not seem to affect potency as compounds 25-26 containing a PEG spacer were equally
potent to compounds 41–42 containing an amide spacer of comparable length (Figure 3A).
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Crystal structures of all inhibitor-virus complexes were obtained and resolved as previously
described, however, similar to the 28-CVA24v complex (pdb-code 6TSD), density was only observed
for the sialic acid units. Thus, decreasing the flexibility of the conjugates by decreasing the spacer length
of 39–42 did not lead to detection of additional electron density from the spacer(s) or core(s).
Pentavalent sialic acid conjugates provide mild stabilization of the CVA24v particle
Antiviral drugs targeting the viral capsid have been suggested to act by stabilization of the virus
particle,40 thereby preventing (or delaying) receptor mediated conformational changes required for
uncoating.41 Pleconaril is one of the most studied capsid-binding molecules and is known to bind to the
hydrophobic pocket of most Enteroviruses where it replaces the hydrophobic pocket factor (a fatty acid),
which stabilizes the virus particle preventing uncoating.42,43 Contrarily, capsid-binding molecules could
also act by destabilizing the virus particle triggering conformational changes, resulting in early uncoating
and inactivation of the virus. To study the effect of the inhibitors on the stability of the CVA24v particle
we performed Particle Stability Thermal Release Assay (PaSTRy).44 We selected the original inhibitor
28 as a representative example of the synthesized compounds. In addition, we selected pleconaril and
sialic acid monosaccharide as controls. The melting temperature (Tm) of the CVA24v particle was
calculated to be 48.40 °C for the proteins (Tm protein) and 50.25 °C for release of the viral genome (Tm
RNA) (Figure 3B). The melting profile is in the same range as for other known Enteroviruses.41
Treatment of CVA24v with the sialic acid monosaccharide shifted the release of the viral genome to
50.96 °C indicating mild thermal stabilization (Figure S5). For the pleconaril control, the melting
temperature shifted to 51.57 °C (Figure S5), which is a relatively weak effect in comparison to other
known Enteroviruses where pleconaril had a thermal stabilizing effect of 3–6 °C.41 Similar to pleconaril
and sialic acid, compound 28 showed a mild stabilizing effect by shifting the Tm for release of the viral
genome to 51.26 °C (Figure 3B). This indicates that the mechanism of inhibition does not involve
destabilization of the viral capsid, and early release of the viral genome, upon compound binding.
Pentavalent sialic acid conjugates inhibit HAdV-37 binding to HCE cells
To study the ability of the pentavalent conjugates to act as general inhibitors of AHC and EKC, we
performed binding experiments using HAdV-37 and HCE cells.23 Compared with untreated virions, the
attachment of 35S-labeled HAdV-37 to HCE cells was significantly reduced when pre-incubated with 28,
46 and 48 (Figure 3C). All tested compounds were significantly more potent than the sialic acid
21
monosaccharide (IC50 1.2 mM)33 and the GD1a glycan (IC50 90.8 µM). Compound 48 (IC50 18 nM) was
roughly 3.7 orders of magnitude more potent than GD1a, while 28 (IC50 34 nM) and 46 (IC50 192 nM)
proved about 2- and 11-fold less effective than 48 respectively.21 These results were in agreement with
previous observations that multivalent sialic acid conjugates with shorter spacers are more potent
against HAdV-37.32,33 The pentavalent conjugates were however not as efficient at inhibiting HAdV-37
binding as reference compound ME0462 (IC50 0.38 nM).33 ME0462 has a nearly perfect fit in the fiber
knob and our previous data indicate that increasing valency might not improve avidity as suggested by
theoretical models.31 The relatively modest avidity gains obtained against CVA24v is likely the result of
CVA24 engaging multiple receptors on the host cells.15,19 Nevertheless, compound 46 and 28 (IC50 177
µM and 248 µM against CVA24v, respectively) proved very efficient at inhibiting HAdV-37 supporting
the prospects of developing a general inhibitor against two of the major causes of EKC and AHC.
The potency of the compounds toward CVA24v may need further improvement. However,
administration is likely possible in millimolar concentrations without adverse side-effects since sialic acid
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is non-toxic and abundantly expressed throughout the human body. Further, repeated administration of
ME0462 in high doses to rabbits did not result in any toxic reactions or adverse side-effects.33
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Spacer length of pentavalent conjugates affects binding to CVA24v and HAdV-37
The mechanism of potency enhancements in multivalent ligand systems are suggested to occur via
chelation, statistical rebinding, cross-linking and aggregation, or via a combination of these mechanisms.
An internal diffusion (bind and slide) mechanism has also been proposed for large systems containing
many (sub)ligands.28 We were interested to study if the mechanism of potency enhancement occurred
via cross-linking and aggregation. To study this, we performed negative staining electron microscopy
experiments with CVA24v and HAdV-37 (Figure 5). Compounds 28, 46, and 48 were selected from the
set of inhibitors as they present a compromise between spacer length and potency toward both CVA24v
and HAdV-37. ME0462 was selected as a control as an aggregation mechanism seems unlikely due to
the short spacers of this compound. Compared with untreated CVA24v (Figure 5, PBS left panel),
ME0462 appears able to break up virion clusters present in the control thus it does not cause
aggregation or cross-linking of viral particles (Figure 5, ME0462 left panel). In contrast, treatment with
28, 46, and 48 causes formation of CVA24v aggregates. Moreover, the extent seems to be related to
spacer length because larger aggregates were observed after treatment with 46 and 28 (long spacers)
in comparison to 48 (Figure 5, 28, 46, and 48 of left panel). This indicates that aggregation may be the
main mechanism of potency enhancement for the inhibitors in regards to CVA24v. It can therefore not
be excluded that the sialic acid density observed in each of the five binding sites the crystal structures
originates from more than one pentavalent molecule cross-linking virus particles.
For HAdV-37, ME0462 showed very little to no aggregation or crosslinking of viral particles in
comparison to untreated virions (Figure 5, PBS and ME0462 of right panel). Particles were also
predominantly monodispersed after treatment with 28 and 48 (Figure 5, 28 and 48 of right panel).
However, substantial aggregation and cross-linking, was observed after treatment with 46 (Figure 5, 46
of right panel). Thus, it is reasonable to speculate that the main mechanism of potency enhancement
for 28 and 48 occurs via a statistical rebinding mechanism by providing a high local concentration of
sialic acid. This can also explain the slightly increased potency of 48 in comparison to 28 as shorter
spacers provide a higher effective local concentration of ligands by a simple reasoning of distance
between unbound ligand and binding site. For 46, it appears that aggregation has major involvement in
the mechanism of potency enhancement. In this particular system aggregation does, however, not seem
to cause additional enhancement in potency in comparison to a statistical rebinding mechanism.
In summary, we find it reasonable to speculate that main mechanism of inhibition for these
inhibitors involves binding to the virion thus blocking the virus from interacting with its cognate
receptor(s) on the cellular surface. This inhibition mechanism can be complemented by e.g. cross-linking
of virus particles were the sialic acid residues and possibly the spacers in one compound bind to two
virus particles.
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Figure 4. Effect of ME0462 and pentavalent sialic acid conjugates on cross-linking and
aggregation of CVA24v and HAdV-37. Left panel shows results of negative staining electron
microscopy for CVA24v and right panel for HAdV-37 in PBS buffer or incubated with trivalent (ME0462)
or pentavalent (28, 48, 46) sialic acid conjugates at a final concentration of compounds at 2 mM. Scale
bars are visible at the bottom left corner for each image and corresponds to 100 nm for images with
CVA24v, and 200 nm for images with HAdV-37.
The sialic acid binding sites of CVA24v and HAdV-37 are different
To further understand the binding of the inhibitors to CVA24v and HAdV-37, and the large differences
in potency we analyzed their respective binding sites. The sialic acid binding sites of each virus are
located in the interface between two protomers of the biological assembly (pentamer and trimer,
respectively). The sialic acid binding sites of HAdV-37 are more recessed and less solvent exposed
because they are located on the inner side of the fiber knobs. They are also more rigid since there is
less mobility in the loops interacting with sialic acid.33 In CVA24v, the receptor-engaging loops show a
higher degree of mobility, and form very shallow binding sites that are solvent exposed. A comparison
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of the total buried surface area (BSA) per sialic acid entity does however show similar values of 160.5
Å2 for CVA24v and 150.1 Å2 for HAdV-37. In HAdV-37, the sialic acid moiety is recognized by direct
contacts with Tyr312, Pro317, and Lys345 and via a water-mediated hydrogen bond to Ser344.33
A
notable difference in the recognition is the presence of a salt bridge between the carboxylate group of
sialic acid and Lys345. In the case of CVA24v, this electrostatic interaction is absent and the carboxylate
is hydrogen bonded to the amide of Ser147 and intramolecularly to the C9 hydroxyl group of sialic acid
(Figure 2D). To fully understand these differences in recognition and their potential contributions to
inhibitor potency further studies are required.
Another underlying factor to explain the potency difference could be that HAdV-37 exclusively rely
on sialic acid glycans for attachment, while CVA24v engage both sialic acid containing glycans and the
co-receptor ICAM-1.19 In summary, several factors seem to contribute in explaining the lower avidity of
the pentavalent conjugates toward CVA24v.
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CONCLUSION
In this study, we have presented the first inhibitors of CVA24v infection and demonstrated the inhibitors
also effectively block HAdV-37 binding to HCE cells. Thus, the synthesized pentavalent sialic acid
conjugates inhibit two of the major causative agents of AHC and EKC, supporting development of
general inhibitors against these highly contagious eye infections. Topical administration directly to the
target tissue in the eye circumvents many of the challenges facing a compound administered orally. The
sialic acid binding sites are located on the virus surface and the inhibitors therefore do not need to
permeate any cell membranes, reducing the risk of off-target effects. Furthermore, systemic exposure
is likely low reducing the risk for toxicity or other adverse effects originating from systemic distribution.
General inhibitors are particularly attractive in this case due to clinical difficulties in distinguishing these
infections and their causative agents. Such inhibitors could therefore be applied upon development of
the first symptoms to alleviate the infections, limit the spread in the general population, and most
importantly to help prevent health care-associated infections of immuno-compromised individuals which
can result in potentially life-threatening conditions.
Supporting information
Materials and methods, supplementary text on docking and calculations, figure S1-5, table S1-3,
chemical synthesis and analytical data, 1H-NMR and 13C-NMR spectra, references (PDF)
Acknowledgements
We thank the Diamond Light Source Ltd. (Didcot, UK) for beamtime and N. Paterson and M. Williams
for their on-site support. The project was funded by a Knut and Alice Wallenberg Foundation grant
(KAW; grant reference 2013.0019, to N. A. and M. E.) and by the Baden-Württemberg foundation,
Glycobiology program (to T. S.).
Author Contributions
E. J., R. C. and W. Q. synthesized all compounds. C. D. A. performed structure-based design. N. M.
and N. C. evaluated the target compounds in cell-based assays. G. Z. performed the crystallography
experiments. N. M. and L. F. carried out the thermal stability experiments. D. H. L. and R. T. carried
out the electron microscopy studies of aggregation. E. J, R. C., M. E., N. A. and T. S., designed the
experiments and analyzed the data together with all other authors. E. J wrote most of the manuscript
with contributions from all other authors. All authors have given approval to the final version of the
manuscript.
The authors declare no competing interests.
Accession Codes
PDB 6TSD
References
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