Ghosh 2008

Glycobiology vol. 19 no. 1 pp.
2–15, 2009
doi:10.1093/glycob/cwn092
Advance Access publication on September 24, 2008
REVIEW
Focus on antivirally active sulfated polysaccharides: From structure–activity analysis
to clinical evaluation
Tuhin Ghosh2,3 , Kausik Chattopadhyay2,3 , Manfred represent one of the richest sources of bioactive compounds,
Marschall4 , Paramita Karmakar3 , Pinaki Mandal3 , and and algae-derived products are increasingly used in medical and
Bimalendu Ray1,3
Downloaded from http://glycob.oxfordjournals.org/ at milner library illinois state university on December 4, 2012
biochemical research (Mayer and Lehmann 2000). Research on
3 Department of Chemistry, Natural Products Laboratory, University of
natural products possessing antiviral activity is mainly focused
Burdwan, WB 713 104, India; and 4 Institute for Clinical and Molecular on low-molecular-weight compounds isolated from plants since
Virology, University of Erlangen-Nuremberg, Erlangen, Germany they can be selected on the basis of their ethno-medicinal use
Received on July 29, 2008; revised on September 19, 2008; accepted on
(Kinghorn 2001). However, some of the aqueous plant extracts
September 19, 2008 used in traditional medicine against viral infection are highly
viscous indicating the additional presence of high-molecular-
In recent years, many compounds having potent antiviral weight compounds. In fact, the first report of the antiviral activ-
activity in cell culture have been detected and some of these ity of high-molecular-weight polysaccharides appeared almost
compounds are currently undergoing either preclinical or 60 years ago (Ginsberg et al. 1947). Seventeen years later, it
clinical evaluation. Among these antiviral substances, nat- was demonstrated that heparin can act as inhibitors of herpes
urally occurring sulfated polysaccharides and those from simplex virus (HSV) (Nahmias and Kibrick 1964). In recent
synthetic origin are noteworthy. Recently, several contro- years, a number of sulfated carbohydrate compounds from ma-
versies over the molecular structures of sulfated polysac- rine algae, cyanobacteria, and animal sources were described
charides, viral glycoproteins, and cell-surface receptors have showing potent inhibitory effects against several human and
been resolved, and many aspects of their antiviral activity animal viruses (Luescher-Mattli 2003; Damonte et al. 2004;
have been elucidated. It has become clear that the antiviral Arad et al. 2006; Pujol et al. 2007). Sulfated polysaccharides
properties of sulfated polysaccharides are not only a sim- of synthetic origin also have antiviral potency (Witvrouw and
ple function of their charge density and chain length but De Clercq 1997). Some of these macromolecules are currently
also their detailed structural features. The in vivo efficacy undergoing clinical evaluation (Kleymann 2005; McReynolds
of these compounds mostly corresponds to their ability to and Garvey-Hague 2007). They have a promising perspective
inhibit the attachment of the virion to the host cell surface to be developed into a novel type of antiviral drugs. This review
although in some cases virucidal activity plays an additional presents an overview of recent preclinical and clinical devel-
role. This review summarizes experimental evidence indi- opments of antivirally active sulfated polysaccharides, with a
cating that sulfated polysaccharides might become increas- particular focus on their structures, structure–activity relation-
ingly important in drug development for the prevention of ships, and mechanisms of action.
sexually transmitted diseases in the near future.
Structural diversity
Keywords: antiviral activity/mechanisms/sulfated
polysaccharides/structural diversity/structure–function Structural analysis of carbohydrates is recognized as one of the
relationship most challenging tasks of glycosciences, given the structural
complexity related to the monosaccharide composition, the vari-
ous isomeric forms, the types of glycosidic linkages, the position
Introduction and distribution of substituents, and the three-dimensional struc-
tures of the molecules (Venkataraman et al. 1999; Nishimura
During the last decade, the number of antivirals approved for et al. 2004; Ashline et al. 2005; Laroy et al. 2006). Extensive
clinical use has been increased from 5 to more than 30 drugs investigation on polysaccharides from various natural sources
(De Clercq 2004). However, as these drugs are not always ef- showed the presence of several classes of highly interesting
ficacious or well-tolerated and drug-resistant virus strains are macromolecules (Painter 1983; Berteau and Mulloy 2003).
rapidly emerging, there is still a great demand for further Structural features of representative polysaccharide classes pos-
drug development including novel modes of action. A very sessing antiviral activities are discussed below.
promising approach is the antiviral screening of products de-
rived from natural sources, such as marine flora and fauna, Sulfated polysaccharides from marine algae
bacteria, fungi, and higher plants. Among them, marine algae Fucan sulfates from marine brown algae usually have com-
plex and heterogeneous structures. Recent studies have also
1 To whom correspondence should be addressed: Tel: +91-342-25-56-56-6; shown that these polysaccharides consistently contain a back-
Fax: +91-342-2634200; e-mail: [email protected] bone of either α-(1→3)-linked or alternating α-(1→3)- and
2 These authors equally contributed to this work. α-(1→4)-linked L-fucopyranosyl residues with sulfate groups

c The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 2
Focus on antivirally active sulfated polysaccharides
Meretrix petechialis (a marine clam) with antiviral activity has

been reported (Amornrut et al. 1999).
Chemically modified compounds

Chemical structures of selected naturally occurring polysac-
charides, such as cellulose, curdlan, dextran, xylan, and others
that have been chemically modified by sulfation, have been re-
viewed (Kennedy and White 1988; Witvrouw and De Clercq
1997). Low-molecular-weight compounds such as highly sul-
fated mannose-containing oligosaccharides and sulfated poly-
mannuronates with a defined structure (supplementary Figure 6)
also have potential antiviral activity. Recently a novel strategy
that uses heparan sulfate (HS) biosynthetic enzymes to gen-
erate biologically active polysaccharides and oligosaccharides
has gained momentum (Munoz et al. 2006; Chen et al. 2007;
Linhardt and Kim 2007; Xu et al. 2008).
Fig. 1. Idealized structure of chondroitin sulfate (CS) chains: CS-A (I), CS-B
(II), CS-C (III), CS-D (IV), and CS-E (V) (Kinoshita et al. 1997; Kinoshita- Structure–activity relationships
Toyoda et al. 2004). The chondroitin sulfate-E isolated from squid cartilage is
a potent inhibitor of HSV-1 infection (Bergefall et al. 2005). Research over the last 20 years has shown that sulfated polysac-
charides possess a broad spectrum of antiviral activities in vitro
(supplementary Tables 1–5). The huge structural diversity of
at position 4 (Daniel et al. 1999; Chevelot et al. 1999; Patankar these macromolecules, however, has given a major hindrance in
et al. 1993; Cumashi et al. 2007) (supplementary Figure 1). This the establishment of their structure–activity relationship. Nev-
regular backbone is frequently masked by different substituents, ertheless, on the basis of the accumulated data, several common
such as monosaccharides (galactose, glucose, mannose, xylose, structural motifs emerge that are highly suggestive to possess
or glucuronic acid), acetyl groups, and/or sulfate esters. The general importance for antiviral activity.
matrix-phase polysaccharides of red seaweeds are linear sulfated
galactans, which contain alternating β-(1→3)-D- and α-(1→4)- Degree of sulfation has a major impact on the antiviral activity
galactopyranosyl residues (Percival and Mcdowell 1967; Rees of polysaccharides
1969; Painter 1983). These galactans differ in the configuration The bulky acidic sulfate substituents that dominate the physical
of the α-linked units (supplementary Figure 2). If the configu- characteristics of polyanions can equally dominate the effects
ration is L-type, the polymer is agaran; in the case of D-type, of such macromolecules in biological systems. Indeed, the im-
it is carrageenan. Different O-linked groups, such as sulfate es- portant parameter for the antiviral activity is the degree of sulfa-
ters, methyl ethers, pyruvate acetal, or monosaccharides, usually tion (DS) of the polymer (i.e., the number of sulfate groups per
mask this regular backbone. Some of the α-galactopyranosyl monosaccharide residue). For a particular class of semisynthetic
units may also occur in the 3,6-anhydro form. The third mem- polysaccharides (e.g., sulfated cyclodextrins), it is well docu-
ber of this class is DL-hybrid galactan sulfate, a polymer in mented that the higher the DS, the better its antiviral potency
which α-linked units can have D- and L-configuration in the (Witvrouw and De Clercq 1997). The general validity of this
same molecule (Stortz and Cerezo 2000). Other polysaccha- finding can be proven with naturally occurring sulfated polysac-
rides having potent in vitro antiviral activities include rhamnan charides of particular classes, such as carrageenans, fucans,
sulfates, spirulans, ulvans (supplementary Figure 3), and xylo- or others, provided they show distinct structural similarities
mannan sulfates (supplementary Figure 4). (Figure 2A). Interestingly, antiviral potencies amongst sulfated
polysaccharides of a given class with similar degree of sul-
Sulfated polysaccharides from animal sources fation, but of different origin, may vary significantly. For ex-
Chondroitin sulfate (CS) and heparin are the two principal ample, the antiviral activity of carrageenan fractions derived
antivirally active macromolecules from animal sources. The from Callophyllis variegate (Rodriguez et al. 2005), Gigartina
precursor polysaccharide for heparin contains repeating β-D- skottsbergi (Carlucci et al. 1997), Gymnogongrus griffithsiae
glucuronic acid (1→4) linked to α-D-glucosamine (Sugahara (Talarico et al. 2004), and Meristiella gelidium (Tischer et al.
et al. 2003; Mulloy 2005). Postpolymerization, enzymes remove 2006), which all possessed a similar sulfate content (approxi-
N-acetyl and replace with N-sulfate, epimerize glucuronate mately 50 mol%), was individually different (Figure 2B). On
residues at its C-5 position, and introduce sulfates at 2 and the other hand, the antiviral IC50 values (herpes simplex virus
6 positions (Kusche-Gullberg and Kjellen 2003). The final re- type 1, HSV-1), fractions obtained from the same algal species
sult of all these transformations is shown in supplementary (Carlucci et al. 1997), correlated well with their charge density
Figure 5. On the other hand, CS chains are composed of glu- (Figure 2A). Although there are clear links between algal species
curonic acid and N-acetylgalactosamine at alternating positions. and polysaccharide classes, the finer structural details differ be-
Several types of CS chains can be distinguished based on the tween certain classes of polysaccharides isolated from different
disaccharide units characterized by sulfate groups at specific species (Lahaye 2001). Against this background, it seems not
positions and the presence of gluco-/iduronic acid residues surprising that IC50 values for various animal viruses can differ
(Figure 1). Recently, a β-D-(1→3) linked galactan sulfate from to markable extents regarding agaroids and carrageenans with
3
T Ghosh et al.
Fig. 3. Antiherpetic activity of sulfated agaran (Chattopadhyay et al. 2008),
sulfated xylomannan (Mandal et al. 2008), sulfated fucan (Mandal et al. 2007),
and sulfated spirulan (Lee et al. 2007) isolated from different marine algae.
mL−1 for HSV-1 and HSV-2), the galactofucan fractions

(21–24% of sulfate and 4–7% of uronic acid) from the same
seaweed showed a strong activity (Ponce et al. 2003) (IC50
values of 0.28–0.87 and 0.52–1.36 µg mL−1 for HSV-1 and
HSV-2, respectively). Similarly, a high uronic acid/low sulfate
fraction (F1M) obtained by anion-exchange chromatography
from galactofucan sulfate of Undaria pinnatifida is less active
(IC50 values of 4.6, 1.0, and 4.0 µg mL−1 for HSV-1, HSV-2,
and human cytomegalovirus [HCMV], respectively), than that
Fig. 2. Antiherpetic activity of (A) polysaccharides having different degrees of of the low uronic acid/high sulfate fraction (F2M) having higher
sulfation but distinct structural similarities and (B) carrageenans with similar
degrees of sulfation but different origins (Carlucci et al. 1997; Rodriguez et al. efficacy (IC50 values of 1.1, 0.1, and 0.5 µg mL−1 , respectively)
2005; Talarico et al. 2004; Tischer et al. 2006). (Hemmingson et al. 2006).
Therefore, with regard to antiviral potency, the polyanionic
nature of a polysaccharide is an absolutely critical factor. In
equal charge density (supplementary Table 1). Again, amongst addition, the type of the anionic group is also important. Sulfates
carrageenans, the λ-type is more effective than κ/ι- and µ/ν- are in many cases required for activity, whereas carboxyl groups
type (Carlucci et al. 1997). The increased activity for the λ-type generally do not promote antiviral activity. Thus, the antiviral
(IC50 lower than 1 µg mL−1 for HSV-1 and HSV-2) compared activity is not merely a function of high charge density, but has
to the κ-carrageenan (IC50 = 1.6–4.1 µg mL−1 ) can be corre- distinct structural specificities.
lated with DS values. For sulfated fucan-containing fractions
(Ec and Ea) of Leathessia difformis, the activity against HSV-1
is proportional to the sulfate content. Recently, it was shown that Specific positioning of sulfates might be important for antiviral
the antiherpetic activity of several other families of polysaccha- activity
rides such as spirulan, agaran, fucan, xylomannan, and their In addition to the well-documented DS dependence, the specific
desulfated and furthersulfated derivatives, is largely depen- position of the sulfate ester group appears to be additionally
dent on the presence of sulfate groups (Adhikari et al. 2006; important for the antiviral activity of sulfated polysaccharides.
Chattopadhyay et al. 2007, 2008; Lee et al. 2007; Mandal et al. This can be demonstrated by using selectively sulfated type
2007, 2008) (Figure 3). As seen from a structural point of view, E chondroitin sulfates (CS-E). Research data showed that CS
a highly charged molecule is more likely to interfere efficiently types A, B, C, and D exhibited either little or no antiherpetic
with electrostatic interactions between the positively charged re- activity (Banfield et al. 1995; Marchetti et al. 2004) while CS-E
gion of a viral glycoprotein and the negatively charged HS chains isolated from squid cartilage exhibited potent antiviral activity
of the cell-surface glycoprotein receptor. In general, sulfated (Bergefall et al. 2005). The specific positioning of sulfates (at
polysaccharides with a sulfate content higher than 20 (mol%) positions 4 and 6) in the predominant CS-E disaccharide unit
have a clear tendency to show an antiviral activity (supplemen- (Figure 1) can explain this surprising inhibitory effect on HSV-1
tary Tables 1 and 2). The distribution of sulfate groups seems infection. The squid cartilage CS-E chains also contain an extra
also important since a low degree of sulfation does not neces- sulfate group at position 3 of glucuronic acid (GlcA) residue.
sarily eliminate the possibility of highly charged zones in the As up to 10% of GlcA residues can be 3-O-sulfated, the CS-E
polysaccharide backbone. chains may possess a domain-like structure with specific po-
Polysaccharides containing high amounts of uronic acid sitioning of trisulfated disaccharide units relative to disulfated
residues, such as alginic acid and pectin, show very little an- units (Kinoshita et al. 1997, 2001). However, one cannot exclude
tiviral activity, which stands in contrast to other polyanionic that in addition to the specific position, the extensive sulfation
compounds. For example, while the uronofucan fractions from of CS-E might contribute to its antiherpetic activity. Another
Adenocystis utricularis (5–12% of sulfate and 22–42% of uronic study (Carlucci et al. 1997) showed that the antiherpetic activity
acid) contain very little antiviral activity (IC50 values >100 µg of natural carrageenans is directly correlated to the amount of
4
(Bergefall et al. 2005). Although the DS of CS-E chains (∼1.7

sulfates/disaccharide) is smaller than that of heparin (∼2.7 sul-
fates/disaccharide), the chain length of CS-E (∼70 kDa) is much
larger than that of heparin (∼12.5 kDa). This illustrates that the
chain length is an important parameter for antiviral potency. The
variation of MW amongst sulfated polysaccharides and their an-
tiviral potency is given in supplementary Tables 1–4.
In some cases, low-molecular-weight-sulfated carbohydrates
can also possess a strong antiviral activity, particularly when the
sulfate content is high. The linear β-(1→4)-polymannuronate-
derived sulfated oligosaccharides (SPMG) with approximately
three sulfates per disaccharide showed size-dependent binding
to human immunodeficiency virus type 1 (HIV-1) glycoprotein
gp120 (Liu, Geng, et al. 2005). However, a certain minimum
Fig. 4. Comparison between the molecular weight and antiviral activity of chain length is required for any of these compounds to show
sulfated carbohydrates. The antiherpetic activity of sulfated agarans of antiviral activity: fragments containing two to five monomeric
Acanthophora spicifera (Duarte et al. 2004), carrageenans of Gigartina residues exhibited low binding capacity to recombinant gp120,
skottsbergi (Carlucci et al. 1997), DL-Hybrid of Gymnogongrus torulosus whereas a 3-fold to 4-fold increase in capacity was observed
(Pujol et al. 2002), and fucans of Leathessia difformis (Feldman et al. 1999)
with specific sulfate content (in mol%).
from hexa- to octasaccharides. This supports the hypothesis that
antiviral activity correlates positively with MW even for low-
molecular-weight compounds. Importantly, the octasaccharide
seems to be the optimum size requirement for inhibiting HIV-1
α-D-galactose 2,6-disulfate residues, supporting the possibility infection, whereas a 19–20 saccharide fragment displays similar
that the specific sulfation of galactose residues might be im- inhibitory potency as native SPMG. These authors used a com-
portant. The results from a more recent study revealed that a petitive inhibition assay together with stoichiometric analysis
3-O-sulfated octasaccharide generated from heparin using an and concluded that sugar chains longer than 15–16 saccharide
enzymatic approach has stronger activity in blocking HSV-1 residues display multivalent interactions with gp120 molecules
infection than that of the 3-OH octasaccharide (Copeland et al. while shorter sugar chains only bind two to three gp120
2008). Therefore, the inhibition of HSV-1 infection requires a molecules. The second example is the HS-mimetic, PI-88,
specific sulfation pattern. which is a mixture of highly sulfated (∼3 sulfates/disaccharides)
Thus, modifications of the sulfation pattern may generate ac- mannose-containing oligosaccharides (supplementary Figure 6)
tivities which normally require higher DS. However, it should (Ferro et al. 2002). Although the DS of PI-88 is very high, be-
be mentioned that there is no general pattern as in which po- cause of its low MW, the inhibitory activity is weak (IC50 =
sition sulfate groups exert the most pronounced antiviral activ- 6 µg mL−1 ) in comparison to large polysaccharides (IC50 =
ity, although it appears that the positioning of the O-sulfates 1 µg mL−1 ). This confirms that a certain minimum chain
on specific sugars is important. Hereby, the density of sulfate length is necessary for the antiviral activity of specific com-
groups on sugars in a particular class of polysaccharide ap- pounds (Nyberg et al. 2004). This concept also implies that a
pears to be mostly critical. Sulfated polysaccharides contain- larger chain is more likely to recognize and interact with nu-
ing 35–60 sulfate groups (in the case of sulfated polysaccha- merous copies of the viral attachment protein(s) and possibly
rides from seaweed) per hundred sugar residues provide the has the ability to cross-link virions. Exceptions from this rule
best antiviral activity (supplementary Tables 1 and 2). In the are some low-molecular-weight compounds, such as heparin-
case of semisynthetic sulfated compounds, polymers containing derived oligosaccharides (DP 10–12), pentosan polysulfates
one and two sulfate groups per monosaccharide residue appear (3 kDa), dextran sulfates (5 kDa), and PI-88 analogs, still pos-
to be optimal (supplementary Table 4). sessing an antiviral activity which is obviously based on other
determinants than MW (Feyzi et al. 1997; Ekblad 2007). An
Molecular weight contributes to antiviral activity alternative way of small-molecular-weight compounds to ex-
press antiviral activity may be the formation of a higher order
Usually an antiviral activity of sulfated polysaccharides corre- structure, such as helixes, that may recognize complementary
lates with the molecular mass of the chain. The higher the aver- regions on target proteins. Albeit, for a great number of antiviral
age molecular weight (MW), the higher is the antiviral activity macromolecules, a certain minimum charge density as well as a
in many cases. When various fractions of semisynthetic glucan certain minimum chain length seems to be essential. Hereby,
sulfates with MWs ranging from 1 to 500 kDa were examined their threshold values are variable and may relate to basic struc-
for their antiviral activity, it was found that the higher MW frac- tural features.
tions were more effective than the lower ones (Witvrouw and
De Clercq 1997). Above ∼100 kDa, no further increase in ac-
tivity was observed. This correlation that is valid for semisyn- Low-molecular-weight compounds inhibit cell-to-cell spread
thetic sulfated polysaccharide can also be extended to natu- of viruses more efficiently
rally occurring sulfated polysaccharides (Figure 4). In some A rapid cell-to-cell spread of viruses is of pivotal importance
cases, the effect of the chain length might overcome that of for the efficiency of viral dissemination in the host organism
charge density. For example, CS-E blocked HSV-1 entry into and thus to establish productive infections in humans. The in-
cells at substantially lower concentrations (IC50 = 0.06 to 0.2 hibition of viral spread may require the penetration of a com-
µg mL−1 ) than standard heparin (IC50 = 1.0–0.8 µg mL−1 ) pound into a narrow intercellular space, and hence the size
5
T Ghosh et al.
correlated with unwarranted cytotoxicity, with longer chains

giving higher cytotoxicity than shorter ones. Moreover, more
hydrophilic chains typically were associated with a lower anti-
HIV-1 activity (18–110 µg mL−1 ) compared to long, branched
or cyclic chains with a higher anti-HIV-1 activity (0.24–0.97 µg
mL−1 ). The most active compound in this series was a lam-
inarapentaoside with a cholesterol aglycone. Terada and co-
workers (2005) studied the HIV-1-inhibiting potency of alky-
lated polysulfated sialic acid derivatives. While the correlation
of the methyl group or linear chains with antiviral activity was
low (no activity with the methyl group, and IC50 = 139 µM
with the linear chain), branched chains possessed a high activity
(IC50 ∼ 1–10 µM). However, a decrease in antiviral activity
was observed for chains containing 26 or more methylene units.
Fig. 5. Selectivity indices (SI = IC50 /CC50 ) of sulfated polysaccharide
isolated from Spirulina platensis with different counter cations (Lee et al. The study by Katsuraya and co-workers (1999) showed that
2001). IC50 (inhibitory concentration 50%): concentration required to reduce sulfated butylated lamina-oligosaccharides containing four glu-
plaque number in Vero cells by 50%. CC50 (cytotoxic concentration 50%): cose residues had only one thirtieth of the anti-HIV activity
concentration required to reduce 50% the number of viable Vero cells after
48 h of incubation with the compounds.
of the sulfated pentasaccharide (IC50 values of 43 and 3.4 µg
mL−1 , respectively). Thus, here again, it is suggestive that a
certain minimum chain length is required for these compounds
to exhibit antiviral activity.
of the compound is frequently a limiting factor. As an exam-
In conclusion, the studies on structure–activity relationships
ple, the low-molecular-weight compounds, PI-88 (∼2.4 kDa)
of sulfated polysaccharides with high antiviral activity demon-
and its analogs, substantially reduce intercellular transmission
strated that the antiviral activity of the sulfated polysaccharides
of HSV-1 (Nyberg et al. 2004; Ekblad 2007). Also in sup-
varies both quantitatively and qualitatively in dependence on
port of this hypothesis, the high-molecular-weight polysaccha-
their structure.
ride compounds heparin (∼15 kDa) and chondroitin sulfate E
(∼70 kDa) are known to affect cell-to-cell spread of HSV-1
only inefficiently, albeit these compounds are potent inhibitors
of infectivity through other mechanisms. Mechanism of action against herpes simplex virus
The entry of HSV into host cells is a complex process initiated
Effect of counter cations
by the specific interaction between host-cell-surface receptors
A recent study has demonstrated that the nature of the counter and viral envelope glycoproteins (Schneider-Schaulies 2000;
cations of anionic sites such as sulfate groups of polyanions Spear 2004; Kleymann 2005; Olofsson and Bergstrom 2005).
play an important role in controlling antiviral activity (Lee et al. All hitherto investigated human herpesviruses, i.e., HSV-1,
2001). For example, Na-spirulan exhibited potent antiherpetic HSV-2, varicella–zoster virus (VZV), human cytomegalovirus
activity but with the replacement of Na+ by another cation, such (HCMV), human herpesvirus (HHV)-7 and HHV-8, except
as Ag+ or Cd2+ , the antiviral potency of this polymer remarkably Epstein–Barr virus (EBV), may utilize cell-surface HS chains
decreased (Figure 5). for primary attachment (WuDunn and Spear 1989; Neyts
et al. 1992; Secchiero et al. 1997; Trybala et al. 2002; Akula
Hydrophobic and hydrogen bonding interactions et al. 2001). Bergefall et al. (2005) analyzed cell lines deficient
Sulfated acylated polysaccharides have been shown to have po- in the expression of glycosaminoglycans (GAGs) and postu-
tent antiviral activity. For example, Yamada and co-workers lated that HSV-1 glycoprotein C (gC) binds to chondroitin sul-
(2000) reported that the low-molecular-weight O-acylated fate (CS, characterized by E disaccharide units) and that the
derivatives of λ- and κ-carrageenans prepared from native CS-E unit is an essential component to function as a HSV-1
polysaccharides increase the anti-HIV-1 activity of the polysac- receptor. More recently, this group showed that chondroitin 4-
charides, while minimizing the native anticoagulant properties. O-sulfotransferase-1 regulates the E disaccharide expression of
It was also observed that the butanoylated derivatives with chondroitin sulfate required for HSV-1 infection (Uyama et al.
0.55–0.70 mol of butanoyl and 1.5 mol of sulfate per monosac- 2006). In the case of HSV-1 and HSV-2, attachment to HS seems
charide residue of λ-carrageenan showed a higher antiviral ac- to be primarily mediated through glycoprotein C (gC) although
tivity (IC50 = 3.9 µg mL−1 ) than dextran sulfate (IC50 = 7.8 µg glycoprotein B (gB) may contribute to this function (Herold
mL−1 ). et al. 1991, 1994; Cheshenko and Herold 2002). Following HS
Sulfated oligosaccharides, having a linear or branched alky- binding, secondary entry receptors are recognized in a cell-type
lated group as aglycon, also possess potent antiviral activity specific manner, before virus entry requires the activity of an-
(Katsuraya et al. 1994, 1999; Ekblad 2007). These compounds other glycoprotein (gD). gD can interact with the herpesvirus
were prepared by glycosylation of oligosaccharides with a vari- entry mediator (HVEM, a member of the tumor necrosis factor
ety of linear, branched, or cyclic aliphatics followed by sulfation receptor family), with nectin-1 and nectin-2 (two related mem-
of the generated glycosides. The lipid chain was attached to cre- bers of the immunoglobulin superfamily) and, additionally, with
ate surface-active agents whereby the alkyl chains coalesced. specific sites in HS generated by certain 3-O-sulfotranferases
All of the sulfated alkyl oligosaccharides evaluated had good (Montgomery et al. 1996; Geraghty et al. 1998; Spear and
anti-HIV-1 activity. Interestingly, the length of the alkyl chain Longnecker 2003; O’Donnell et al. 2006; Xu et al. 2005). The
6
gD interactions possibly trigger conformational changes in vi-

ral gB and gH/gL components thus initializing fusion between
the viral lipid envelope and cell plasma membrane (Spear 2004;
Perez-Romero et al. 2005). Subsequently, viral capsids and tegu-
ments proteins are released into the cytoplasm of the host cell.
Alternative pathways of HSV-1 entry into the cell can occur in
some cell-types through endocytosis. Endocytosed HSV-1 fuses
with the endosomal membrane instead of the cytoplasmic mem-
brane (Nicola et al. 2003). By using a second entry pathway, viral
glycoproteins can mediate HSV-1 entry via the apical cellular
surface in the form of a cell-to-cell spread, i.e., the movement
of virions across the narrow space between infected and adja-
cent uninfected cells (Cocchi et al. 2000; Kusche-Gullberg and
Kjellen 2003).
The receptor-binding site of viral glycoproteins
HSV gC (designated gC1 and gC2 for HSV-1 and HSV-2, re-
spectively) is a type 1 membrane glycoprotein that contains 511
(gC1) or 480 (gC2) amino acids and exhibits 65% identity in
the primary amino acid sequences between gC1 and gC2 (Swain
et al. 1985). Although the receptor-binding motifs of gC have
not been completely elucidated yet, it is well documented that
gC1 has nine sites for N-linked oligosaccharides and numerous
sites for O-linked glycans. The latter are clustered at the N-
terminal part of the protein, which make this region structurally
similar to mucins. The mucin-like region is not present in gC2.
The eight cysteins in gC1 form four disulfide bonds (Rux et al.
1996). Clusters of basic and hydrophobic amino acids located
between residues 129 and 160 of gC1 (Trybala et al. 1994;
Mardberg et al. 2001, 2002) as well as the mucin-like re-
gion (amino acids 33–123) of this protein (Tal-Singer et al.
1995) were identified as important for HSV-1 attachment to
cell-surface HS/CS.
HSV-1 gB1 consists of 904 amino acids, and approximately
85% of the sequence is homologous to its HSV-2 counterpart
(Heldwein et al. 2006). Most of the variability between gB1 and
gB2 is seen in a lysine-rich region (amino acids 68–76), which
is also responsible for binding to HS (Laquerre et al. 1998). gB1
Fig. 6. Ribbon diagram of a single gB1 protomer. (Reprinted with permission
occurs as a trimer with each of the monomers divided in five from Heldwein et al. (2006) (http://www.sciencemag.org) Copyright 2006,
distinctive domains: I base, II middle, III core, IV crown, and V AAAS.)
arm (Heldwein et al. 2006; Figure 6). The results from a more
recent study suggest that specific hydrophobic/aromatic amino
acids from domain I are important for the fusogenic activity of (Shukla et al. 1999; Xia et al. 2002; Chen et al. 2003; Tiwari et al.
gB (Hannah et al. 2007). 2005; Xu et al. 2005 O’Donnell et al. 2006). A gD binding oc-
tasaccharide motif (Liu et al. 2002) and a gC binding N-sulfated
Cell-surface glycosaminoglycan receptors dodecasaccharide motif (Feyzi et al. 1997) were characterized
Heparan sulfate, an endogenous ligand for many viral proteins, confirming that HSV-1 utilizes a unique HS sequence for its
is initially synthesized as a copolymer of alternating (1→4)- entry. The structural motifs of the glycoepitopes on HS chains
linked β-D-glucuronic acid and N-acetylated α-D-glucosamine, are given in Figure 7. Recent results from cell-based assays re-
and then undergoes various modifications in the Golgi appa- vealed that the inhibition of HSV-1 infection requires a unique
ratus (Esko and Lindahl 2001; Lindahl et al. 1998). These sulfation moiety (Copeland et al. 2008).
modifications include N-deacetylation and N-sulfation of glu-
cosamine, C5 epimerization of glucuronic acid to form iduronic Sulfated polysaccharide acts through complex processes
acid residues, 2-O-sulfation of iduronic and glucuronic acid An attractive concept is that sulfated polysaccharides act as an-
residues, as well as 6-O-sulfation and 3-O-sulfation of glu- tiviral agents in cell culture due to the fact that these charged
cosamine residue (Gorsi and Stringer 2007). HS chains can polymers may mimic HS chains on cell-surface proteogly-
provide specific binding sites for various proteins, exemplified cans and thus block viral attachment by competitive inhibition.
by binding of HSV-1 gB that depends on the presence of one or A novel approach to inhibiting HSV-1 infection by targeting
more 6-O-sulfate and 2-O-sulfate groups, and gD that requires the gD-mediated membrane fusion step has recently been de-
a HS chain modified by 3-O-sulfotransferases isoforms II–VI scribed (Copeland et al. 2008). This inhibition was achieved by
7
T Ghosh et al.
Fig. 7. Chemical structure of disaccharides (VI and VII) relevant for binding to glycoprotein C (gC) and glycoprotein D (gD) of HSV-1.
using a unique 3-O-sulfated octasaccharide, which was gener- of sulfated polysaccharides onto interferon production with the
ated from heparin using an enzymatic approach. The results of consequence of a broad antiviral effect.
this study also demonstrate that the inhibition of HSV-1 can be
blocked by saturating the viral envelope glycoprotein gD using
a small molecule of defined structure. The antiherpetic proper-
Mechanism of action against human immunodeficiency
ties of sulfated polysaccharides may depend not only on their
virus type 1
charge density but also on the characteristics of their uncharged
portions which may be involved in hydrophobic and hydrogen HIV-1 infection is a multistep process beginning with the at-
bonding interactions. Mardberg and co-workers (2001) reported tachment of the virion to cell surfaces and entry into lym-
that hydrophobic interactions, in addition to electrostatic forces, phoid target cells. In several cell types, including CD4+ helper
are decisive for the CS as well as HS binding to viral glyco- T cells, macrophages, and dendritic cells (Balzarini and Van
protein gC. It has been suggested that sulfated carbohydrates Damme 2007; McReynolds and Garvey-Hague 2007; Nikolic
can elicit profound conformational changes in hydrophobic re- et al. 2007), the virus can establish productive or persistent
gions of proteins and cause disassembly of complex proteins infection. During virus spread, HSV-1 virons are attached via
consisting of subunits (Sedlak and Antalik 1998). Therefore, their envelope gp120 glycoprotein to the CD4 surface protein
the interaction of the methyl groups of fucoidan with the hy- of T-lymphocytes in conjunction with a chemokine (CXCR4 or
drophobic pocket of HSV-1 gC seems to be important in the CCR5) coreceptor. The formation of a ternary complex between
binding of the polysaccharide to the viral glycoprotein. While gp120 and the receptor/coreceptor triggers the fusion between
sulfated oligosaccharide chains can bind to and block the viral the virion and cytoplasmic membrane, a step mediated by viral
attachment/receptor-binding proteins, it was speculated that for gp41. Fusion is followed by the internalization of the virus and
this class of viral inhibitors the hydrophobic group might addi- particularly the viral genome into the nucleus of the target cell
tionally insert into the viral lipid envelope (Uryu et al. 1992). (Moore and Stevenson 2000). Besides CD4 and chemokine re-
This might lead to a destabilization and irreversible inactivation ceptors, attachment determinants of the cell surface play a role in
of the virion. viral infection. For example, HIV-1 can bind to cell-surface hep-
Finally, in addition to the polysaccharide-mediated antiviral aran sulfate proteoglycans via the polybasic V3 loop of gp120
effects directed to the cell surface (viral receptor binding, en- (Roderiquez et al. 1995). This is a sequential process in which
try, fusion), a second type of effects may play a role, i.e., the HS first binds through a high-affinity, selective interaction with
induction of intracellular events contributing to the antiviral ac- the V3 loop on gp120, followed by a second, lower affinity
tivity of sulfated polysaccharides. As the binding of a number of interaction with the conserved chemokine coreceptor region of
known polysaccharides to cell-surface receptors can induce in- gp120 (Moulard et al. 2000). Other studies have shown that HS
tracellular signaling pathways, this second type of effects should can compensate for low levels of CD4 in macrophages, thus
be additionally taken into consideration. As an example, the an- regulating HIV-1 infection in these cells (Saphire et al. 2001). A
ticytomegaloviral effect of spirulan-like polysaccharides was recent study by de Parseval and co-workers (2005) highlighted a
demonstrated to be composed of these two antiviral activities, single highly conserved amino acid in the V3 loop, Arg 298, and
i.e., an inhibition of HCMV entry on the one side in addition its importance in binding to both HS and CCR5. The interesting
to the induction of intracellular anti-HCMV effects on the other and perhaps most significant, aspect of the findings in this study
side (Rechter et al. 2006). Due to the fact that the replication was that a specific sulfation motif of HS was preferred, 6-O-
efficiency of most viruses is dependent on specific intracellular sulfation, indicating that random sulfation or negative charges
signaling pathways, the inhibition or the induction of particular were not sufficient to yield a strong binding event between the
signaling of surface-binding polysaccharides can provide a sig- virus and the host cell.
nificant part of the overall antiviral activity. One explanation for One of the important questions regarding viral envelope pro-
such intracellularly produced activity is the stimulatory effect teins is what are their finer structural details and recent X-ray
8
treated with i.p. administration of dextrin 2-sulfate, there was

a significant decrease in viral load (Shaunak et al. 1998). Data
from a more recent clinical study suggest that dextran sulfate
is absorbed rapidly in humans after oral administration. Curd-
lan sulfate (CRDS), another potent anti-HIV-1 polysaccharide
was found to produce a marked dose-related increase in CD4+
lymphocytes (Gordon et al. 1994, 1997). This increase mainly
returned to baseline after 24 h. In the later study, these authors
found that after 21 days of intravenous administration at three
dose levels of CRDS in HIV-1- and HCMV-infected individuals,
12 out of 21 patients tested were negative for HCMV.
Low-molecular-weight anionic compounds generally seem
to possess a better bioavailability than high-molecular-weight
compounds. Indeed, the smaller size of the highly sulfated
mannose-containing oligosaccharides (PI-88) may account for
their in vivo biological activity (Lee et al. 2006). SPMG, the
alginate-derived sulfated oligosaccharides that are in phase II
clinical trial as virucides is another example (Liu, Geng, et al.
2005; Hui et al. 2006).
Side effects
The major undesirable side effect of sulfated polysaccharides is
their well-known anticoagulant activity. This adverse effect can
be avoided by selecting sulfated polymers, such as fucoidan,
galactan, spirulan, xylomannan, and heteroglycan, that have no
discernible anticoagulant activity at the therapeutically admin-
istrable doses (Hayashi et al. 1996; Ghosh et al. 2004; Adhikari
et al. 2006; Chattopadhyay et al. 2007, 2008; Mandal et al.
Fig. 8. Structure of HIV-1 gp120 including the functionally highly relevant
V3 loop. As depicted, the viral surface would be at the top and the target cell at
2007, 2008). Among anionic polysaccharides, soluble cellulose
the bottom of the figure. (Reprinted with permission from Huang et al. (2005) acetate phthalate, a potential candidate as microbicide, had a
(http://www.sciencemag.org). Copyright 2005, AAAS.) minimal effect on plasma coagulation (Neurath, Strick, Li, et al.
2002).
Cytotoxicity at the site of administration is a common prob-
lem with the use of low-molecular-weight compounds with viru-
crystallographic studies have provided a complete picture of the
cidal effects. This may be explained by the unwarranted deter-
gp120 structure (Chen et al. 2005; Huang et al. 2007). Huang
gent effect of these compounds toward both, viral and cellular
and co-workers (2005) reported the structure of HIV-1 gp120,
membranes. The adverse interaction of polysaccharide com-
complexed to CD4 and the X5 antibody, whereby the V3 loop
pounds with other medications is rather unlikely due to the
structure was maintained (Figure 8). This was an important
relatively large size of the polysaccharides and their inefficient
finding, as it confirmed the role of the V3 loop in HIV-1 entry,
resorption into the body. For this reason, topical administra-
providing critical structural information about a region that is
tion of polysaccharide compounds seems generally much more
prone to mutation.
promising than oral/parenteral administration. Indeed, the most
Numerous investigations on the mechanism of action of sul-
active sulfated carbohydrates are able to block in vitro viral in-
fated polysaccharides have been conducted for HIV-1. Sulfated
fection at a concentration as low as 0.01 µg mL−1 , whereas no
polysaccharides can block viral entry into host cells by inter-
toxicity to host cells has been detected at concentrations up to
fering with the attachment of virus to the cell-surface receptors.
1–2.5 mg mL−1 (supplementary Tables 1, 2, and 4). Therefore,
These macromolecules may exert their anti-HIV-1 activity by
the selectivity index and hence the antiviral potency of these
shielding off the positively charged amino acids present in the
compounds are very high.
viral envelope glycoprotein gp120 (Harrop and Rider et al. 1998;
Moulard et al. 2000). gp120 has several highly basic regions,
most notably, the V3 loop, which can interact with polyanionic Drug resistance
regions of host-cell-surface molecules (Vives et al. 2005). Sulfated polysaccharides may inhibit the attachment of viruses
with target molecules on the cell surface. The viral attachment
peptides are highly conserved regions within rather variable
Clinical applications scaffolds of viral surface glycoproteins. These peptides are only
poorly subject to alterations by the natural antigenic drift of
Many researchers have conducted in vivo studies on the bioavail- viruses. Likewise, they are not expected to represent frequent
ability of sulfated polysaccharides. It has been observed that the sites of drug-induced resistance mutation. Sulfated polysaccha-
in vivo effectiveness of oral administration of dextran sulfate to rides that are directed toward these target peptides are therefore
HIV-1 was disappointing (Flexner et al. 1991). But, in an open preferred candidates for antiviral drug development. Moreover,
phase I/II dose-escalation study, in which six AIDS patients were sulfated carbohydrates showed in vitro antiviral activity against
9
T Ghosh et al.
virus mutants resistant to nucleoside analogs (Adhikari et al. sulfated polysaccharide exhibits a broad spectrum of activity
2006; Chattopadhyay et al. 2007; Mandal et al. 2007, 2008). against Chlamydia trachomatis, Gardnerella vaginalis, Grad-
Further research is certainly needed to address other open ques- nerella vaginalis, Neisseria gonnorhoeae, and human papilo-
tions of polysaccharide-induced resistance of viruses. mavirus (McReynolds and Garvey-Hague 2007).
The phase III clinical trial of candidate microbicides, Carra-
Virucidal activity guard, and cellulose sulfate did not show that these compounds
are effective in preventing HIV transmission (Cohen 2008;
The virucidal activity of compounds is highly relevant for their
Population Council 2008). Therefore, for the next generation of
antiviral activity in vivo. For example, λ-carrageenan showed
microbicide candidates, more emphasis must be placed on the
an irreversible, virucidal mode of action against HSV-1 proba-
selection of only the most potent compounds prior to commenc-
bly due to its high affinity binding to the virion and exhibited
ing a clinical trial process that moves inexorably toward phase III
antiherpetic activity in laboratory mice (Carlucci et al. 1997).
efficacy trials. In this respect, the R5 SHIV vaginal challenge
Other such polysaccharides include dextrin-x-sulfate (DxS, ML
model in nonhuman primates (Lederman et al. 2004; Veazey
Laboratories, UK), cellulose acetate phthalate (CAP), galactan
et al. 2005) provides the most relevant and stringent available
sulfates from Grateloupia longifolia and Grateloupia filicina
test of how a product might perform in humans. This model is a
(Wang et al. 2007) and a linear β-(1→3) and (1→4) linked
vitally important tool when rational choices between candidates
sulfated glucan from Avena sativa bran (Wang et al. 2008). In
and formulations must be made. Had the current generation of
comparison with other antivirally active sulfated polysaccha-
microbicide products undergone challenge studies in the pri-
rides, heparin does not inactivate HSV-1 virions and does not
mate model, fewer might have failed in efficacy trials in humans
show an interaction with virion components in an irreversible,
(Shattock and Doms 2002). Next generation concept now in
virucidal way.
or approaching clinical trials offer improved prospects for ef-
ficacy. The most plausible approach involves a combination of
Polysaccharides as microbicides several drugs, preferentially targeting different steps in the vi-
Microbicides are compounds that applied vaginally or rec- ral infection process. Because sulfated polysaccharides are safe
tally and protect the user from sexually transmitted infec- and acceptable (Bollen et al. 2008; Kilmarx et al. 2008; van der
tions. Polyanions are one of the most studied microbicides. Straten et al. 2008), development of several second-generation
This group of potential microbicides includes cellulose sulfate, combination formulation based on first generation lead candi-
dextran/dextrin sulfates, carrageenan, and cellulose acetate ph- dates may be more effective (Liu, Lu, et al. 2005; Brache et al.
thalate. 2007; Gantlett et al. 2007; Klasse et al. 2008). PC-815, a novel
Carraguard R
, the Population Council’s lead candidate micro- combination microbicide containing carrageenan and the nu-
bicide, is a sulfated polysaccharide containing a mixture of ι- cleoside reverse transcriptase inhibitor MIV-150, is one such
and κ-carrageenan (Coggins et al. 2000; Kilmarx et al. 2006). second-generation microbicide. Pharmacological testing con-
In contrast to detergents and pH-buffering agents (two other ducted in vitro indicated that PC-815’s activity against HIV-1
classes of microbicides), Carraguard has not demonstrated con- is significantly higher than the activity of Carraguard alone
traceptive properties, a possible advantage in situations where (Fernandez-Romero et al. 2007). Because Carraguard and MIV-
protection against pathogens, but not contraception, is desired. 150 have different mechanisms of action against HIV-1 when
DxS is a synthetic sulfated polysaccharide (∼ 20 kDa), whose formulated into PC-815, there is an additive effect in activity.
antiviral activity is distinct from related dextran sulfate (Javan Similarly, the combination of CADA with CAP resulted in a
et al. 1997; Shaunak et al. 1994, 2003). These macromolecules synergistic inhibition of HIV-1 and SIV infection (Vermeire
have shown varying levels of protection against a simian human et al. 2008). How these inhibitors are applied may also be criti-
immunodeficiency virus 89.6 (SHIV-89.6) vaginal challenge in cal, with sustained release formulations and vaginal ring deliv-
the rhesus macaque model (Veazey et al. 2005). A recent study ery systems (Woolfson et al. 2000, 2006; Malcom et al. 2005)
showed that DxS are active against R5 virus in cellular and now becoming a high priority.
tissue models (Fletcher et al. 2006). AusAm Biotechnologies is
considering phase I clinical trials in the herpes indication with
DES-6, a sulfated dextran derivative (Kleymann 2005). Conclusions
A number of studies have demonstrated the efficiency of CAP
as a topical microbicide (Orozco-Topete et al. 1997; Gyotoku Recent structural studies showed that sulfated polysaccharides
et al. 1999; Kawamura et al. 2000; Neurath et al. 2001; Neurath, represent a group of macromolecules possessing more proper-
Strick, Jiang, et al. 2002; Neurath, Strick, Li, et al. 2002). CAP ties in common than previously thought. These macromolecules
is not soluble at pH <5.5, normal for microbicide target sites. have a variety of antiviral properties, particularly due to their
Micronized CAP is the only candidate topical microbicide with ability to imitate patterns of sulfate substitution on GAGs present
the capacity to remove rapidly by adsorption from physiological in cell membranes. Because the structures of sulfated polysac-
fluids HIV-1 of both the X4 and R5 biotypes and is likely to charides are complex and heterogeneous and as many stud-
prevent virus contact with target cells. The interaction between ies of antiviral activity were carried out using relatively crude
micronized CAP and HIV-1 leads to rapid virus inactivation. In polysaccharide preparations, it is presently not easy to deter-
contrast to sulfated polysaccharides, CAP had a minimal effect mine the overall relationship between activity and structure.
on plasma coagulation. However, the results of many studies demonstrate that the an-
Finally, cellulose sulfate (Ushercell, Polydex Pharmaceuti- tiviral activity of sulfated polysaccharides is not merely a func-
cals, Ontario, Canada) was tolerated in phase I and II studies tion of high charge density and chain length but also distinct
(Balzarini and Van Damme 2007; Nikolic et al. 2007). This structural characteristics. Generally, high-molecular-weight
10
polysaccharides appear to possess the most pronounced in- ficiency virus; SP, polysaccharides containing fractions derived
hibitory activity toward viral receptor binding and entry. With from Spirulina platensis; SPMG, sulfated polymannuronate;
respect to the bioavailability and the cell-to-cell spread of viri- Tat, transactivator of transcription; TNF, tumor necrosis factor;
ons, however, low-molecular-weight compounds appear to be VZV, varicella–zoster virus.
superior to their high-molecular-weight counterparts. Investiga-
tions of the mechanism of action revealed that viral adhesion,
transport, and entry into host cells are complex processes medi- References
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Glycobiology vol. 19 no. 1 pp.

Meretrix petechialis (a marine clam) with antiviral activity has

Chemically modiﬁed compounds

mL−1 for HSV-1 and HSV-2), the galactofucan fractions

(Bergefall et al. 2005). Although the DS of CS-E chains (∼1.7

correlated with unwarranted cytotoxicity, with longer chains

gD interactions possibly trigger conformational changes in vi-

treated with i.p. administration of dextrin 2-sulfate, there was

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