Keywords
pentosan polysulfate - SARS-CoV-2 - COVID-19 - antiviral agent
Introduction
The enveloped, positive sense RNA virus, SARS-CoV-2 (severe acute respiratory syndrome
coronavirus 2), belonging to the Coronaviridae, is responsible for the coronavirus disease 2019 (COVID-19) pandemic and causes serious
clinical morbidities that are consistent with the onset of SARS.[1] By late 2021, the total number of COVID-19 deaths worldwide stood at over 5 million,
but this is probably an underestimate owing to the difficulties of collecting accurate
data globally. Despite the significant contribution of vaccines to preventing much
severe morbidity and mortality caused by SARS-CoV-2 infection, there remains an urgent
need to develop additional drugs to treat or prevent the infection. COVID-19 is a
disease which continues to occur due to the waning of immunity, the infection of immunosuppressed
patients, or those with underlying illness, as well as the emergence of mutations
and the resulting breakthrough infections. Several drugs, marketed for other therapeutic
indications, have been re-purposed to treat COVID-19 patients and antiviral strategies
that include treatment with remdesivir or convalescent plasma have received emergency
approval.[2]
[3] Despite promising results, the use of such treatments is limited, owing to their
expense and, because they can only be delivered intravenously. Additional treatments
are therefore required and, indeed, during the writing of this article, the first
orally available antiviral drug against COVID, molnupiravir, was approved for use
in the United Kingdom.[4]
[5]
One existing family of drugs which have been re-purposed for treatment of COVID-19
is heparins. Heparins are a class of polydisperse, linear, sulfated polysaccharides
of animal origin. Besides their well-known anticoagulant properties, heparins have
been shown to inhibit cell invasion by SARS CoV-2,[6]
[7] SARS CoV-1,[8]
[9] influenza H5N1,[10] as well as several flaviviruses, including Dengue and Yellow Fever viruses.[11] Heparin can also influence the ability of the infected cell to survive, as evinced
by a study of the survival of cells infected by Zika virus.[12] The activity of heparin against SARS-CoV-2 has been established using several in
vitro experimental models and its efficacy appears to depend largely on its interactions
with the spike protein 1 receptor binding domain (S1-RBD).[6]
[7]
[13]
Regarding the clinical use of heparin, a systematic review and meta-analysis of the
association of anticoagulant status and mortality in COVID-19 patients was conducted
encompassing 29 articles that had been published before January 2021. The study concluded
that both therapeutic and prophylactic anticoagulant regimes reduced in-hospital mortality
compared with untreated patients.[14] Anticoagulant therapy has been recommended in the prophylaxis of thromboembolism
in COVID patients with respiratory infection and reduced mobility.[15] Nevertheless, the occurrence of bleeding complications and the documented risk of
heparin-induced thrombocytopenia (HIT) after prolonged exposure both limit the use
of heparin in patients having high incidence of venous thromboembolism, particularly
for high-dose administration.
The proposed mechanism of inhibition by heparin involves competition for the binding
of the SARS-CoV-2 spike protein (S1) with endogenous cell-surface heparan sulfate
(HS), a co-receptor which serves as an anchor, and to facilitate the conformational
change that enables viral binding to angiotensin-converting enzyme 2 (ACE2).[6]
[7] The high negative charge density of heparin, a close structural analogue of HS,
favors interaction with predominantly positively charged amino acid residues of S1
(R346, R355, N354, R357, K444, and R466).[6]
[16] The results from several studies have suggested that other sulfated molecules and
highly charged polyelectrolytes could also provide potential targets for the design
of inhibitors against the entry of viruses.[17]
[18]
Pentosan polysulfate (PPS) is a chemically sulfated derivative of pentosan, a plant-derived
xylan that has been approved for the treatment of bladder pain and discomfort in interstitial
cystitis (ema.europa.eu/en/medicines/human/EPAR/elmiron) and could represent an alternative
to heparin. PPS is produced by the exhaustive chemical O-sulfation of hardwood glucuronoxylan
whose backbone comprises a linear polymer of D-xylopyranose (Xyl) units linked β (1–4)
to each other and branched with 4-O-methyl-D-glucuronic acid (MGA) residues linked
α (1–2) to the xylose backbone[19] ([Fig. 1A]). PPS has poor anticoagulant activity and is known for its anti-inflammatory activities
and affinity for the cytokines, interleukin (IL)-4, IL-5 and IL-13, resulting in similar
anti-inflammatory efficacy to topical steroids in a guinea pig model of allergic rhinitis,
following intranasal administration.[20] Indeed, aerosol administration could be an attractive approach for upper airway
drug treatments, thereby enabling the relatively low bioavailability of PPS after
oral administration[21]
[22]
[23] to be overcome.
Fig. 1
(A) Major structural signatures of PPS. MGA: branching 4-O-methyl-2,3-di-O-sulfated glucuronic
acid, Ac: acetyl group. (B) Low-field and high-field proton NMR spectral regions of PPS and its fractions. PPS,
pentosan polysulfate.
In the present study, a sample of PPS (API) was fractionated according to its charge
density and molecular dimension using anion exchange chromatography. The parent polysaccharide
and six fractions were characterized in terms of their molecular weight distribution,
as well as by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass
spectrometry (LC-MS) analysis. These were all then evaluated for their ability to
bind the S1-RBD and to inhibit virus invasion in comparison with both unfractionated
heparin (UFH) and its low molecular-weight-heparin (LMWH) counterpart. The reduced
anticoagulant potency of PPS and its fractions compared with heparin, alongside its
well-tolerated oral administration even at high dose (300 mg daily),[24] suggests that oral administration or, even better, aerosol administration of PPS
might provide an effective and safe prophylactic treatment of COVID-19 disease.
Methods
Preparation of PPS Fractions
Native PPS (Bene PharmaChem GmbH, Germany) was fractionated on a DEAE Sephacel column
applying aqueous NaCl (0.6–2.0 M, stepwise elution) as eluent. PPS fractions were
precipitated by the addition of a twofold excess of ethanol and storing at −30°C overnight.
Samples were collected by centrifugation (3,500 rpm, 10 minutes), washed with 70%
ethanol, and dried in vacuo at 40°C.
NMR Characterization of PPS and PPS Fractions
1H-NMR and heteronuclear single quantum coherence (HSQC) NMR spectra were acquired
using a Bruker Avance 500 NEO instrument (Bruker, Karlsruhe, Germany), equipped with
5 mm cryoprobe. Ca. 40 mg of samples were weighed and dissolved in 0.5 mL of deuterated
phosphate buffer solution (PBS), 0.15 mM, pH 7.1. Proton spectra were recorded at
303 K, with presaturation of solvent during relaxation delay. Recording and integration
of quantitative HSQC spectra as well as quantification of the monosaccharide building
blocks were performed according to the published study.[19]
Molecular Weight of PPS and PPS Fractions
The molecular weight distributions of PPS and PPS-derived fractions were performed
on a Viscotek 305 HPLC system (Viscotek, Houston, United States) equipped with a triple-detector
array exploiting simultaneous application of refraction index (RI) detector, viscometer
and right-angle laser light-scattering (RALLS), employing an adaptation of the published
method.[25]
[26] The applied RI increment (dn/dc) was found to be 0.093 mL/g and was used to convert the RI response to concentration.[19]
LC-MS Characterization of PPS and PPS Fractions
Analyses by LC-MS of unfractionated PPS and its fractions were performed by ion pair
reversed phase chromatography coupled with high-resolution mass spectrometry as previously
described.[19] Briefly, the separation of PPS oligomers was achieved on a HPLC 1100 (Agilent, Santa
Clara, United States) system equipped with a high-resolution ESI-FTICR mass spectrometer
Solarix 7T (Bruker Daltonics, Billerica, Massachusetts, United States), using a Kinetex
2.6 μm C18 column (100 × 2.1 mm I.D., Phenomenex, Torrance, California, United States)
and dibutylamine acetate as ion pairing agent (eluent A and B containing 10 mM DBA
and 10 mM acetic acid in water and methanol, respectively). All reagents were of LC-MS
grade. The separation was achieved using a multistep gradient at 0.1 mL/min flow rate:
the solvent composition was held at 35% B for the first 3 minutes, then increased
to 55% B over 2 minutes, and to 75% B over another 35 minutes, where it was held at
75% for 20 minutes; afterwards, the content of eluent B was increased to 98% in 30 minutes,
where it was held for 10 minutes to elute the longest components. Finally, it was
returned to 35% B over 2 minutes, and held for the last 23 minutes for equilibrating
the chromatographic column before the next run. The following ESI-MS (electrospray
ionization mass spectrometry) parameters were used: capillary voltage, 3.2 kV; nebulizer
gas pressure, 1 bar; drying gas, 3.7 mL/min, and 180°C. The mass spectra acquisition
was performed in m/z 200 to 3,000 range with time domain 1M and ion source accumulation of 300 milliseconds.
Injection volume was 3 μL for both 5 mg/mL unfractionated PPS and 2.5 mg/mL fractions.
The fraction, Fr.2.0M, was not analyzed by LC-MS due to poor chromatographic separation
and the poor ionization of high-molecular-weight oligosaccharides, which results in
low MS sensitivity.
Dynamic Light Scattering
Dynamic light scattering (DLS) experiments were performed using a Zetasizer Nano ZS
instrument (Malvern Panalytical, Malvern, United Kingdom) operating at 633 nm with
backscatter detection at 173°. The protein solution (2 μM) was allowed to equilibrate
with or without the addition of salt at the measurement temperature of 25 °C before
starting acquisition. To analyze the S1-RBD/PPS complex size, the solutions were obtained
by mixing S1-RBD with unfractionated and fractionated PPS solutions in 20 mM HEPES
buffer with salt at different protein ligand molar ratios. The complexes were transferred
to a disposable cell at room temperature and analyzed 2 minutes post-mixing. Data
were analyzed using Zetasizer software version 7.11 (Malvern Panalytical, Malvern,
United Kingdom) and each value corresponds to the average of three separate measurements.
Circular Dichroism
Circular dichroism (CD) spectra data were collected at 25°C on a Jasco J-1500 instrument
(JASCO, Great Dunmow, United Kingdom) equipped with a Peltier unit-controlled cell
holder. The spectra in the far-ultraviolet (far-UV) region (200–250 nm) were recorded
using a quartz cell (Hellma, Southend-on-Sea, United Kingdom) with a path length of
0.1 cm, a response time of 1 second, a scan speed of 20 nm/min, and bandwidth of 0.5 nm,
and each spectrum was the average of five scans. To address the conformational state
of the S1-RBD domain in HEPES buffer, CD data were acquired on S1-RBD (4–10 µM) samples
just after reconstitution with de-ionized water as a control and after buffer exchange
(HEPES buffer, pH 7.4 with NaCl 200 mM). To analyze the conformational changes upon
PPS complex formation, the S1-RBD solutions were incubated with unfractionated and
fractionated PPS solutions in HEPES buffer at different protein ligand molar ratios
and CD spectra were recorded immediately.
Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) was used to characterize the binding affinities
between PPS fractions and the S1-RBD domain. The experiments were performed at 25°C
using a MicroCal PEAQ-ITC (Malvern Ltd., Malvern, United Kingdom). Both protein and
ligand were prepared in PBS or HEPES buffer pH 7.2. Briefly, SARS-CoV-2-RBD protein
(Sino Biological, China) was first reconstituted with MilliQ filtered water and then,
to remove the excess of trehalose, the buffer was exchanged with PBS or 20 mM HEPES
pH 7.2, NaCl 60 mM using an Amicon Ultra concentrator (10 kDa filter, 0.5 mL). Each
lyophilized PPS fraction was initially resuspended with 500 µL of the same buffer.
The protein and ligand stock solutions were than diluted to different concentrations
for ITC analysis with PPS or HEPES buffer and 140 or 200 mM salt. For all the experiments,
S1-RBD was taken in the cell (2–3 μM) and PPS fractions in the syringe (30 μM). Each
ITC experiment consisted of 20 injections of 2 μL each with an initial delay of 180 seconds
between injections, and a stirring rate of 500 rpm. To estimate the thermodynamic
parameters (K
D, ΔH, and ΔS), the data were fitted using the MicroCal analysis software.
Vero Cell Culture and Assays
Vero cells were plated at 2.5 × 105 cells/well in 24-well plates in EMEM supplemented with 10% fetal bovine serum (complete
medium). Twenty-four hours later, three different PPS protocol treatments were adopted:
-
Cell treatment: Cells were incubated with serial dilutions of PPS in 250 µL of complete medium for
30 minutes and then 50 µL of supernatant containing 50 plaque-forming units (PFU)
of SARS-CoV-2 isolate (GISAID accession ID: EPI_ISL_413489) were added.[6]
-
Virus treatment: 50 PFU of SARS-CoV-2 in 50 µL of complete medium were incubated with compound serial
dilutions of PPS for 30 minutes at 37°C and then added to Vero cells in a final volume
of 300 µL.
-
Cell + virus treatment: 50 PFU of SARS-CoV-2 in 50 µL of complete medium were incubated with compound serial
dilutions and cells were incubated with serial dilutions of PPS within the same range
for 30 minutes at 37°C in 250 µL. After 30 minutes of incubation at 37°C, the virus-containing
supernatant was added to the PPS-treated cells.
In all three PPS protocol treatments, the incubation was then extended for 1 hour
at 37°C. Then, supernatants were discarded and 500 µL of 1% methylcellulose overlay
dissolved in medium containing 1% of fetal bovine serum was added to each well. After
3 days, cells were fixed using a 6% (v/v) formaldehyde:phosphate-buffered saline solution
and stained with 1% (w/v) crystal violet (Sigma-Aldrich, Italy) in 70% (v/v) methanol
(Sigma-Aldrich, Italy). The plaques were counted under a stereoscopic microscope (SMZ-1500,
Nikon).
Statistical Analysis
Prism GraphPad software v. 9.0 (www.graphpad.com) was used for the statistical analyses. Comparison among groups was performed using
the one-way analysis of variance (ANOVA). The mean of each treatment column was compared
with the control column (Nil; no treatment) applying the Bonferroni correction. The
symbol **** indicates a p-value <0.0001, *** indicates a p-value <0.001, and ** indicates a p-value <0.01.
Coagulation Assays
All fractions were stored frozen (−20°C) at 1 mg/mL in Aqua-Dest. Heparin (A3004,0001,
196 IU/mg; Applichem, Darmstadt, Germany) and LMWH (enoxaparin, 100 mg/mL, Sanofi-Aventis,
Frankfurt, Germany) were stored according to the manufacturer's instructions for use.
Dilutions in human pool plasma were used with the following concentrations (first
dilution step to 100 µg/mL in 0.9% NaCl) (n = 6): 0; 3.125; 6.25; 12.5; 25; and 50 µg/mL. The activated partial thromboplastin
time (aPTT; SyntASil TM; reference range: 25–38 seconds) and anti-Xa activity (Liquid
anti-Xa TM; reference range 0 IU/mL) were measured with commercially available assays
according to the manufacturer's instructions (Werfen, Munich, Germany) and were analyzed
on the ACL TOP analyzer (Werfen, Munich, Germany).
PPS Structural Optimization and Molecular Docking Simulations of PPS-SARS-CoV-2 S1-RBD
Domain
A model comprising the linear hexasaccharide portion of PPS was represented by the
xylohexasaccharide: 2,3,4-trisulfo-Xyl-β(1–4)-[2,3-disulfo-Xyl]5-OH. Two limit conformations of this xylohexasaccharide, characterized by all 2,3-disulfo-Xyl
residues in 1C4 or 4C1 conformations, were built. In each 2,3-disulfo-Xyl residue, the conformation of the
sulfate groups was manually adjusted to reduce the steric hindrance considering their
orientation as axial or equatorial corresponding to the 1C4 or 4C1 chairs. In both models, the ɸ
i
/ψ
i
glycosidic backbone conformation was also manually adjusted before the final energy
minimization step, to reduce the steric hindrance of the glycan chain. The final conformations
of these two xylohexasaccharides having Xyl residues in 1C4 and 4C1 were energy-minimized using the Maestro 12.7 Macromodel 13.1 software and molecular
mechanic Amber* force-field (Suite 2021–1, Schrödinger Inc. San Diego) ([Supplementary Fig. S5], available in the online version). The nonbonded distance cut-offs were set as 20.0,
8.0, and 4.0 Å for electrostatic, Van der Waals, and hydrogen bond potential energy
interactions, respectively. The minimization procedure involved 10,000 steps (default
energy minimization algorithm) or a gradient threshold 1.0 × 10–3 KJ mol−1 Å−1, before the docking stage. Only the xylohexasaccharides in 1C4 (see the Results section) was submitted to molecular docking.
The hexasaccharide was docked on the surface of the S1-RBD (PDB ID 6M0J) targeting
the sequence of positively charged patches R346, R355, K356, R357, which present good
accessibility when the S1-RBD is in the “up” conformation in the trimeric protein
S (PDB ID 6VSB). All the hexasaccharide torsional degrees of freedom were adjusted
during automatic docking, except for the glycosidic dihedrals ɸ
i
/ψ
i
(i = 1, 5), while the S1-RBD was set as rigid. The docking grid was set as Lx
= Ly
= 80, Lz
= 120 points, while the center of the grid was: (CM
x
, CM
y
, CM
z
) = (−40.272, 25.851, 21.266). The grid spacing was 0.375 Å (default). The software
Autodock 4.2 was used for the automatic docking. The Lamarckian genetic algorithm
4.2 was used for the docking in combination with the following set of parameters:
number of runs = 100, population size = 2,000, maximum number of energy evaluations = 2.5 × 107, maximum number of generations = 2.7 × 105. After the search procedure, the obtained poses of the ligand–receptor complex were
clustered (tolerance root-mean-square deviation < 2.0 Å) and each clusters characterized
by the lowest binding energy (Autodock score function) and highest population (number
of poses for cluster) were selected.
Results
Isolation and Structural Characterization of PPS Fractions
To correlate the structural properties of PPS with their ability to bind the spike
protein and inhibit virus propagation, PPS was fractionated by anion exchange chromatography
and fractions were characterized by light scattering, NMR and LC-MS.
The molecular weights of PPS and its fractions were obtained by size exclusion chromatography
coupled with a triple detector array (HP-SEC-TDA), consisting of light scattering,
refractive index, and viscometer, following a similar procedure to that adopted for
UFH and LMWHs.[25]
[26] The advantage of this method compared with classical approaches is that it does
not require column calibration with reference standards possessing the same chemical
structure as the samples to be analyzed, nor do they need to exhibit well-defined
molecular weight and narrow polydispersity. The accuracy of such relative measurements
is therefore affected by the quality of the standards employed. The distinct elution
times of PPS and its fractions reflects their different molecular weight and molecular
weight distributions ([Supplementary Fig. S1], available in the online version). The M
w (weight average molecular weight), M
n (number average molecular weight), polydispersity D (expressed as M
w/M
n), R
h, and η (dL/g) values obtained for the samples are reported in [Table 1]. The M
w values of the fractions ranged between 3.7 and 17.3 kDa, corresponding to an average
chain length of 11 to 50 monosaccharide units.
Table 1
Properties of PPS and its fractions; the results refer to the mean values of duplicate
injections
|
Sample
|
M
w (kDa)
|
M
n (kDa)
|
D (M
w/M
n)
|
R
h
[a]
|
η[b] (dL/g)
|
|
(PPS)
|
5.7
|
4.4
|
1.3
|
1.6
|
0.01
|
|
(Fr. 1.0M)
|
3.7
|
3.6
|
1.0
|
1.3
|
0.04
|
|
(Fr. 1.1M)
|
5.3
|
5.3
|
1.0
|
1.6
|
0.05
|
|
(Fr. 1.2M)
|
7.0
|
6.9
|
1.0
|
1.9
|
0.06
|
|
(Fr. 1.3M)
|
9.2
|
9.1
|
1.0
|
2.3
|
0.08
|
|
(Fr. 1.4M)
|
11.8
|
11.7
|
1.0
|
2.7
|
0.10
|
|
(Fr. 2.0M)
|
17.3
|
17.0
|
1.0
|
3.4
|
0.15
|
Abbreviation: PPS, pentosan polysulfate.
a Hydrodynamic radius.
b Intrinsic viscosity.
PPS and its fractions, except for fraction Fr. 2.0M, with the highest molecular weight,
were analyzed by LC-MS. The LC-MS profiles with the identified components are shown
in [Fig. 2] with the m/z data and retention times of the components. Some additional minor structures identified
are also reported in [Supplementary Fig. S2] (available in the online version). Overall, the fractions differ from each other
principally in the length of oligosaccharides, whereas all the structural groups (e.g.,
sulfate groups, MGA, and acetyl moieties) identified in PPS are homogeneously distributed
in each fraction, as confirmed by NMR analysis described below.
Fig. 2 TIC profiles of PPS and PPS fractions with the identified components. Xyl: xylose
repeating unit, MGA: branching 4-O-methyl-glucuronic acid, S: sulfate group, Ac: acetyl
group, Py: pyridine moiety. PPS, pentosan polysulfate; TIC, total ion current.
All the fractions contain linear O-sulfated oligomers Xyl
n
S2n+1 with one unsubstituted hydroxyl group at C1 of the reducing end (RE) ([Fig. 1A]) as prevalent components ([Fig. 2]). Oligosaccharides with two unsubstituted hydroxyl groups (Xyl
n
S2n
) are detected only in trace amounts, confirming essentially the complete sulfation
of the internal chains ([Supplementary Fig. S2], available in the online version). Minor oligosaccharides with an additional 14
Da mass shift Xyl
n
S2n+1 generally accompany the most abundant Xyl
n
S2n+1 species and show multiple peaks in the LC-MS profile, most likely representing various
positional isomers related to the presence of L-rhamnose instead of D-xylose.
For all fractions, ion exchange chromatography-based fractionation led to co-elution
of regular Xyl
n
S2n+1 oligosaccharides with minor dehydrated Xyl
n
S2n+1-H2O oligomers, these latter formed by the loss of a water molecule from the RE, for
Xyl
n
S2n
-H2O, containing a C2–C3 double bond at the RE ([Supplementary Table S1], available in the online version). The co-elution of shorter dehydrated oligomers
with Xyl
n
S2n+1 suggests that the former is better retained by the ion exchange column, most likely
because of their higher polarity. An inverse trend is observed for pyridine-containing
oligomers. The Xyl
n
S2n+1Py envelope is slightly shifted to longer chains ([Fig. 2]) that may be explained by charge compensation between a negatively charged sulfate
and positively charged nitrogen atom of a pyridine moiety ([Fig. 1A]). Interestingly, MGA-branched structures, with or without 3-O-acetylation (Xyl
n
MGA1S2n+1Ac1 and Xyl
n
MGA1S2n+2, [Fig. 1A]), are also characterized by slightly longer chains than the regular Xyl
n
S2n+1 oligomers. Their co-elution might be associated with weaker retention of MGA-branched
structures on the anion exchange column due to steric effects. As previously reported,
MGA-containing structures often exhibit multiple peaks of positional isomers.[19]
Qualitative and quantitative fingerprints of monosaccharide composition of PPS and
its fractions were obtained by 1H and HSQC-NMR spectroscopy, according to methods recently described ([Fig. 1B] and [Supplementary Table S1], available in the online version).[19] The monosaccharide composition was determined by 1H NMR ([Table 2]). The data provided quantitative information concerning some structural features
of PPS which can be related directly to the xylan source (e.g., MGA content) and its
preparation process, such as minor modifications at the RE of PPS chains (e.g., through
formation of double bonds, pyridine derivatives, and possible methylation as well
as residual acetylation). The RE signal of α-xylose at 5.38 ppm decreases with the
increase in molecular weight, in agreement with data obtained by HP-SEC-TDA and LC-MS.
ΔXylred (CHO) was calculated from the integral of the H3 signal at 6.78 ppm, while
the level of Xyl3Ac-2MGA was calculated from the acetyl signal at 2.31 ppm. Finally,
the degree of acetylation was determined by calculating the ratio of the sum of acetyl
groups between 2.35 and 2.1 ppm and the sum of xylose-related signals.
Table 2
Monosaccharide compositional analysis by 1H NMR (molar percentage)
|
Residue
|
PPS
|
Fr. 1.0M
|
Fr. 1.1M
|
Fr. 1.2M
|
Fr. 1.3M
|
Fr. 1.4M
|
Fr. 2.0M
|
|
Xyl + XylNR
|
67.5
|
68.9
|
73.6
|
75.5
|
76.4
|
76.9
|
82.4
|
|
Xylα
|
5.0
|
4.7
|
2.6
|
2.0
|
1.5
|
1.3
|
1.0
|
|
Xyl3Ac-2MGA
|
2.3
|
2.6
|
2.7
|
2.7
|
2.6
|
2.6
|
1.9
|
|
ΔXyl
|
0.6
|
0.4
|
0.3
|
0.3
|
0.2
|
0.1
|
0.2
|
|
ΔXylred(CHO)
|
1.1
|
1.0
|
1.3
|
1.0
|
0.8
|
0.5
|
0.6
|
|
Xylα(Py) + Xylβ(Py)
|
1.4
|
1.3
|
0.6
|
0.5
|
0.3
|
0.2
|
0.2
|
|
MGA-(Xyl3Ac)
|
2.2
|
2.2
|
2.5
|
2.7
|
2.6
|
2.5
|
1.9
|
|
MGA-(Xyl)
|
1.1
|
1.2
|
0.8
|
0.9
|
0.9
|
0.7
|
0.7
|
|
MGA*-(Xyl)
|
1.0
|
0.8
|
0.3
|
0.3
|
0.2
|
0.2
|
0.6
|
|
Degree of acetylation
|
4.1
|
4.8
|
5.1
|
5.2
|
5.2
|
5.5
|
4.5
|
Abbreviation: PPS, pentosan polysulfate.
The advantage of the quantitative 2D-NMR method[27]
[28] consists of the enhancement of signal dispersion compared with the mono-dimensional
spectra, which enables better resolution of additional minor signals of PPS. The molar
percentages of each residue ([Supplementary Table S1], available in the online version) were calculated from the abundance of corresponding
cross-peak volumes normalized to the sum of all the cross-peak volumes.[19] Both mono- and bidimensional techniques indicated the structural similarity of all
PPS fractions, which differ mainly by the quantity of their reducing and non-reducing
residues ([Table 2] and [Supplementary Table S1] [available in the online version]).
ITC Determination of SARS-CoV-2 S1-RBD Binding to PPS
ITC is a sensitive technique for detecting biomolecular interactions by measuring
the heat absorbed or released upon binding. ITC does not require immobilization or
labeling of the partners and can be performed in solution, thereby measuring the affinity
of binding molecules in their native state. In addition, ITC experiments provide not
only the parameters relating to binding, such as binding constant (K
D) and stoichiometry (N), but also the enthalpy and entropy contributions of that binding (ΔH, ΔS) providing information concerning the nature and magnitude of the forces driving
the binding. To evaluate the possible correlation between the binding affinity and
the PPS chain length, ITC experiments were performed to address the interaction between
the S1-RBD and PPS fractions with different average chain lengths. ITC experiments
were first performed in PBS that mimics the pH, osmolarity, and ion concentration
of human fluids. The ITC profiles of S1-RBD titrated with PPS and its fractions were
fitted using the two binding-site models, involving two independents rather than sequential
binding sites corresponding to a strong primary binding site and a nonspecific, secondary
binding site ([Supplementary Table S2], available in the online version). To minimize nonspecific binding, we performed
the experiments increasing the salt concentration. At higher ionic strength (500 mM),
the added salt led to full screening of the electrostatic interaction between PPS
chains and S1-RBD with no evident heat changes in the ITC profiles. Furthermore, an
increase of the salt concentration from 140 to 200 mM resulted in nonreproducible
ITC titrations, despite the protein sample and experimental conditions remaining unchanged
during repeated measurements. We therefore changed the buffer solution from PBS to
HEPES, which is another widely used buffer in biological studies, suitable for ITC
experiments owing to its low ionization enthalpy. The state and conformational stability
of the protein in HEPES buffer were further confirmed by DLS measurements and CD spectroscopy
([Supplementary Fig. S3], available in the online version).
ITC thermograms resulting from the titration of S1-RBD in HEPES buffer using the same
PPS fractions that were employed in the in vitro antiviral activity measurements are
shown in [Fig. 3]. The ITC profiles showed exothermic heat of binding that could be fitted using a
one-site binding site model ([Table 3]). The binding affinity was found to increase with the increasing length of ligand,
as the apparent dissociation constant decreased as ligand molecular weight increased
(from a K
D of 8.410−7 to 7.42 × 10−8 M). For all fractions, N values were in the range of 0.18 to 0.24, indicating that four to five molecules
of S1-RBD were bound to a single molecule of PPS. A similar binding stoichiometry
had already been observed for PF4 and FGF1, which form multicomponent complexes in
the presence of heparin.[29]
[30] It can be hypothesized that one PPS chain is sandwiched between two dimers of S1-RBD
by the negative charges present on both sides of the oligosaccharide. However, further
experiments are necessary to confirm this hypothesis.
Fig. 3 Representative profiles of ITC titration at pH 7.2 of S1-RBD domain with fractions:
(A) Fr-1.0 M, (B) Fr-1.2 M, and (C) Fr-1.4 M PPS in 20 mM HEPES buffer and 200 mM of salt. The upper panels show the
raw data (heat pulse for every injection). The bottom panels represent integrated
areas per mole of injected ligand as a function of molar ratio. The solid line is the best fit to the experimental data using a single-site binding model. ITC,
isothermal titration calorimetry.
Table 3
Thermodynamic parameters obtained from ITC titrations of PPS fractions into S1-RBD
domain using a single-site binding model
|
MW (KDa)
|
N (sites)
|
K
D (M)
|
∆H (kcal/mol)
|
∆G (kcal/mol)
|
−T∆S (kcal/mol)
|
|
PPS[a]
|
5.7
|
0.20 ± 0.01
|
6.13 × 10−7 ± 3.96 × 10−7
|
−2.20 ± 1.45
|
−8.48
|
−6.28
|
|
Fr-1.0M
|
3.7
|
0.24 ± 0.05
|
8.41 × 10−7 ± 5.36 × 10−7
|
−0.97 ± 0.34
|
−8.29
|
−7.31
|
|
Fr-1.1M
|
5.3
|
0.18 ± 0.11
|
4.33 × 10−7 ± 3.48 × 10−7
|
−0.96 ± 0.74
|
−8.68
|
−7.72
|
|
Fr-1.2M
|
7.0
|
0.24 ± 0.05
|
2.85 × 10−7 ± 1.58 × 10−7
|
−5.78 ± 1.80
|
−8.93
|
−3.15
|
|
Fr-1.3M
|
9.2
|
0.23 ± 0.04
|
1.84 × 10−7 ± 1.00 × 10−7
|
−4.02 ± 0.99
|
−9.19
|
−5.17
|
|
Fr-1.4M
|
11.8
|
0.22 ± 0.04
|
1.61 × 10−7 ± 1.07 × 10−7
|
−4.64 ± 1.34
|
−9.27
|
−4.63
|
|
Fr-2.0M
|
17.3
|
0.22 ± 0.02
|
7.42 × 10−8 ± 3.69 × 10−8
|
−5.24 ± 0.70
|
−9.73
|
−4.49
|
Abbreviations: ITC, isothermal titration calorimetry; PPS, pentosan polysulfate.
Note: Titration was performed in 20 mM HEPES buffer pH 7.4 and 200 mM NaCl (140 mM NaCl).
a 140 mM NaCl.
The binding to the S1-RBD resulted in negative binding free energy that became slightly
more favorable at increasing ligand molecular weights (−8.29 to −9.73 kcal/mol). All
interactions had both favorable enthalpy (−0.96 to −5.78 kcal/mol) and entropy (−3.15
to −7.72 kcal/mol) terms. The negative enthalpic contribution to binding was mainly
due to the formation of strong ionic interactions, in addition to hydrogen bonds and
van der Waals interactions between the side chains of basic residues on the S1-RBD
surface and the sulfate groups of the ligand. The favorable entropic contribution
was presumably due to the higher release of water molecules to bulk upon ligand and
target interaction.
In contrast with PPS fractions, titration experiments performed using the PPS mixture
as a ligand showed no interactions at 200 mM NaCl, consistent with a low affinity
of binding (K
D in the mM range), which could not be detected with ITC measurements ([Supplementary Fig. S4], available in the online version). However, calorimetric titrations performed decreasing
the salt concentration at 140 mM resulted in a binding isotherm with a K
D of 6.13 × 10−7 ± 3.96 × 10−7 M and with binding that was enthalpically (∆H = − 2.20 kcal/mol) as well as entropically (−T∆S = − 6.28 kcal/mol) driven. The resulting binding stoichiometry was similar to that
found for the fractions, where 4–5 S1-RBD molecules bind one molecule of PPS. To confirm
that high salt concentrations screened any electrostatic interactions between PPS
and S1-RBD, an ITC titration was performed, lowering the salt concentration to 60 mM
([Supplementary Fig. S4], available in the online version). The fitted data yielded a lower dissociation
constant (K
D: 1.64 × 10−7 ± 0.47 × 10−7 M), suggesting that electrostatic interactions contribute significantly to the affinity
between the PPS and the S1-RBD domain.
Structural Characterization of SARS-CoV-2 S1-RBD upon Binding to PPS
Since no significant changes were observed in the hydrodynamic radius of the S1-RBD
in complex with the PPS chains, we investigated whether alterations in the S1-RBD
structure accompany the binding. Far-UV CD spectroscopy that is sensitive to conformational
changes in proteins upon ligand binding was employed. Specifically, the CD signal
in the far-UV (200–250 nm) provides information concerning changes in the proportions
of secondary structural elements (α-helices, β-sheet, and unstructured regions) that
could take place following ligand binding. The far-UV CD spectra acquired on S1-RBD
at increasing ligand concentration reported only slight changes of secondary structure,
without highlighting any trend related to increased concentrations of PPS or its fractions
([Table 4]). In agreement with the DLS data, no increase in antiparallel β-sheet content was
measured, which can indicate aggregation, nor was an increase in the level of signal
amplification required (which would have been indicative of scattering arising from
aggregation). These results suggest that the various ligands tested induce broadly
similar conformations in the protein and are consistent with the interpretation that
no significant aggregation was associated with the binding.
Table 4
Conformational changes in S1-RBD upon addition of PPS or PPS fractions, determined
by far UV-CD
|
P:L ratio
|
Helix
|
Antiparallel
|
Parallel
|
Turn
|
Others
|
NRMSD
|
|
PPS
|
0.0
|
4.3
|
35
|
0.5
|
12.1
|
48.0
|
0.044
|
|
0.2
|
4.5
|
37.7
|
0
|
12
|
45.9
|
0.026
|
|
0.3
|
4.3
|
38
|
0
|
13.5
|
44.2
|
0.025
|
|
0.9
|
7.3
|
34.5
|
2.8
|
12.3
|
43.2
|
0.020
|
|
Fr-1.0M
|
0.0
|
4.5
|
35
|
2.5
|
12.5
|
45.6
|
0.033
|
|
0.2
|
4.4
|
35.9
|
3.1
|
10.3
|
46.2
|
0.036
|
|
0.3
|
4.1
|
36.4
|
0
|
13.4
|
46.1
|
0.026
|
|
0.9
|
7
|
36.7
|
0
|
13.2
|
43.2
|
0.022
|
|
Fr-1.2M
|
0.0
|
6.4
|
37.7
|
0
|
15.2
|
40.7
|
0.044
|
|
0.2
|
3.8
|
39.3
|
0.3
|
9.5
|
47.0
|
0.039
|
|
0.3
|
8.5
|
37.5
|
0.6
|
9.3
|
44.1
|
0.035
|
|
0.9
|
3.8
|
39.4
|
0
|
14
|
42.8
|
0.039
|
|
Fr-1.4M
|
0.0
|
7.5
|
37.6
|
2.3
|
10.6
|
42.2
|
0.034
|
|
0.2
|
4.7
|
34.9
|
2.7
|
12.1
|
45.6
|
0.032
|
|
0.3
|
4.1
|
35.4
|
1.6
|
14.1
|
44.7
|
0.026
|
|
0.9
|
4.1
|
35
|
3.1
|
11.4
|
46.3
|
0.034
|
Abbreviations: NRMSD, normalized root-mean-square deviation; PPS, pentosan polysulfate;
UV-CD, ultraviolet circular dichroism.
PPS Binding-Site Analysis
The determination of the possible contacts between a 2,3-disulfated polyxylan oligosaccharide
and the S1-RBD of the SARS-CoV-2 was performed by molecular docking simulation, in
which the hexasaccharide 2,3,4-trisulfo-Xyl-β(1–4)-[2,3-disulfo-Xyl]5-OH was docked onto the convex surface of the S1-RBD (receptor). The conformation
of 2,3-disulfo-xylan residues was set in the 1C4 conformation as suggested by 3JH-H couplings, nuclear Overhauser effect contacts, and molecular modeling analysis ([Supplementary Fig. S5], available in the online version). The xylohexasaccharide model was built in two
limit conformations, setting all the xylose (Xyl) residues in 1C4 and 4C1 chair forms. After energy minimization, the xylohexasaccharide with residues in 1C4 showed the lowest potential energy (−5,318.81 kJ/mol) compared with the 4C1 conformation (−5,281.10 kJ/mol). Interestingly, the 1C4 xylohexasaccharide allows greater distances between the two sulfate groups located
at 2-O- and 3-O- of each Xyl residues ([Supplementary Fig. S5], available in the online version). The observed inversion of conformation of the
xylose pyranoside ring from 4C1 to 1C4, resulting from repulsion between the negatively charged sulfate groups, has also
been observed for the internal chain residues of 2–3-disulfo-Xylo-oligosaccharides[31] and the glucuronic acid residue of chondroitin, following full chemical O-sulfation.[32]
The targeted site on the S1-RBD was identified by the series of amino acids that are
characterized by predominantly positively charged side chains: R346, N354, R355, R357,
K444, R466, which remain accessible from the solvent when the S1-RBD is in the “up”
conformation on the trimeric S protein. This region has been identified previously
as the preferred molecular recognition site toward glycosaminoglycans.[6] The docking simulation was set up centering the grid box on the Cα of K356 and taking care to leave suitable space for the ligand to be adjusted on
the surface of the S1-RBD, considering all possible relative orientations between
the ligand and the receptor that were explored in the automatic search procedure.
The docked solutions were clustered (tolerance < 2.0 Å), and the clusters ranked by
the predicted binding energy (Autodock 4.2 score function). Clusters of docking solutions
were then selected considering the binding energy and the population of the clusters.
Analysis of the docking solutions suggested weak specificity in the molecular recognition
between the hexasaccharide and the S1-RBD of SARS-CoV-2. In fact, 22 clusters of poses
(populated by from 1 to 6 poses) presented binding energies between −3.5 and −1 kcal/mol.
Interestingly, the lowest binding energy structures (−3.5 kcal/mol) correspond to
two different clusters of docking poses. These two sets of docking solutions are reported
in [Fig. 4], showing contacts between the amino acids of the site I identified on the S1-RBD
and the sulfate groups of the hexasaccharide. These solutions are located at the edge
of the β-sheet that includes the site I sequence (N354, R355, K356, R357), and correspond
to the shallow groove delimited by the short helix (C336 and R346) from one side and
the loop (P463-S469) to the other side ([Fig. 4]). In this case both docking solutions present comparable contact distances (2.7–4.0 Å)
between the residues of the site I core (N354, R355, K356, R357) and the nearest sulfate
groups of the hexasaccharide.
Fig. 4 Xylohexasaccharide 2,3,4-trisulfo-Xyl-β(1–4)-[2,3-disulfo-Xyl]5-OH docked on the S1-RBD of the SARS-Cov-2. The representations correspond to the
two preferred docking solutions (A and B panels), decomposed in terms of binding energy, but showing different molecular recognition
patterns (RMSD > 2.0 Å). Distance between the sulfate groups of the ligand and the
side chains of selected amino acids are underlined by dashed lines and reported in
Å. The S1-RBD is shown in white ribbon. The amino acids that belong to the site I
of the S1-RBD6, are drawn in green, blue, and red tubes, represent carbon, nitrogen, and oxygen
atoms, respectively. The concave ACE2-binding surface is oriented to the top of the
image. The approximate positions of C336, P463, and S469 are underlined to help the
reader to locate the helix (C336-R346) and the loop (P463-S469) delimiting the recognition
for linear poly-sulfated glycans (see the text). RMSD, root-mean-square deviation.
SARS-CoV-2 Viral Plaque-Forming Assays and S1-RBD Binding
It has been established that the anionic, linear cell surface polysaccharide, HS,
is an endogenous receptor for several pathogenic viruses,[8]
[9]
[11] including SARS-CoV-2[6]
[7] and that heparin, a closely related, negatively charged polysaccharide with structural
similarities to HS, can inhibit this interaction.[6]
[7]
[10] It has been observed for several protein interactions that other sulfated polysaccharides,
including those obtained by chemical sulfation of naturally occurring materials, can
emulate heparin and HS, to induce biological effects.[33]
[34] The possibility that PPS, which is itself a semi-synthetic product made by the chemical
sulfation of a naturally occurring plant xylan and may possess similar inhibitory
activities was therefore investigated.
PPS is an established and approved treatment for cystitis, which can be administered
orally, and shows very low toxicity even at high dose (300 mg/day).[24] It is also reported to exhibit potentially advantageous anti-inflammatory properties.[20] This alternative potential inhibitor was therefore explored using an established
cell-based model of SARS CoV-2 virus invasion and its potency was found to be similar
to UFH ([Fig. 5A]). It was found that PPS was able to inhibit viral invasion in experiments involving
all three forms of addition; PPS added to cells (cell), to virus (virus), and when
added to both before being mixed (virus + cell) as shown in [Fig. 5A]. All three independent experimental approaches highlighted an inhibitory capacity
of PPS at least equal to, if not greater than, that of heparin (UFH) on a weight per
volume basis.
Fig. 5 PPS inhibition of SARS-CoV-2 infection. (A) Vero cells were treated with PPS and heparin (both at 100 μg/mL) prior to infection
with 50 PFU of SARS-CoV-2 or virus-containing supernatant (50 PFU) was treated with
the PPS or heparin. In addition, both cells and virus were treated with PPS or heparin
as described in the Methods and Materials sections. (B) Dose response when Vero cells were treated with PPS serial dilution prior to infection
with 50 PFU of SARS-CoV-2, virus-containing supernatant. (C) Dose response of PPS and PPS fraction-mediated inhibition of SARS-CoV-2 viral invasion
of Vero cells. 50 PFU were treated with the PPS serial dilution, both cells and virus
were treated with PPS. (D) The PPS-mediated inhibition of SARS-CoV-2 viral invasion of Vero cells (100 μg/mL)
compared with heparin and LMWH. One-way ANOVA with Bonferroni correction was used
to determine the p-values. Nil represents no treatment. ANOVA, analysis of variance; LMWH, low-molecular-weight
heparin; PPS, pentosan polysulfate.
The PPS fractions showed some variation in activity. When compared on a molar basis,
a direct M
W dependence was observed, the higher molecular weight fraction (Fr-1.4M; M
w 11.8 kDa) being the most active ([Fig. 5C]). However, attributing this with confidence to any particular structural difference
is more difficult because the fractions have broadly similar compositions ([Table 2]). The inhibitory efficacy of PPS, UFH, and LMWH (enoxaparin) were also compared
on a weight per volume basis ([Fig. 5D]). It is clear that PPS is an inhibitor as effective as UFH, when added to cells,
on a weight per volume basis (achieving ca. 60% inhibition compared with the no inhibitor
control), and is more potent than LMWH (which achieved ca. 25% inhibition ([Fig. 5D]), which has a closely comparable molecular weight ([Table 1]). Additionally, both PPS and its fractions inhibited viral infection in a dose-dependent
manner (in the range 12.5–200 µg/mL) ([Fig. 5B, C]).
PPS and Fractions Exhibit Reduced Anticoagulant Activity Compared with Heparin
A key advantage of PPS is that it exhibits reduced anticoagulant potential ([Supplementary Table S3], available in the online version) which is an important consideration, since high
doses may be required in any use as an antiviral agent. Moreover, PPS is less likely
to induce bleeding complications. The conventional approach to avoiding the most serious
side effects of prolonged treatment with heparin, such as heparin- HIT, is to employ
LMWH, and a similar strategy could be envisaged in attempting to inhibit SARS-CoV-2.
The anticoagulant activity of PPS and derived fractions was measured as aPTT activity
in comparison with heparin and enoxaparin. As expected, a steady increase of anticoagulation
activity with higher molecular weight PPS fractions was observed in the aPTT assay,
but always clearly less than heparin.
Discussion and Conclusion
Discussion and Conclusion
Almost 2 years after the onset of the SARS-CoV-2 pandemic, most countries are still
to reach the high levels of vaccination coverage that is necessary to achieve herd
immunity. This is compounded by the gradual loss of immunity among those who have
already been vaccinated or those who have recovered from an earlier infection, and
this gradual loss of protection has also been linked to frailty, obesity, and deprivation.[35] The only therapeutic options available currently to treat the early symptoms of
coronavirus are injectable antiviral drugs and it was only at the beginning of November
2021 that the first oral antiviral drug (molnupiravir) was approved for use in the
United Kingdom. There is, therefore, an on-going need to develop additional treatments.
While heparins, the established family of anticoagulant polysaccharides, have shown
promise in clinical use,[36]
[37] reservations remain concerning their direct use as antiviral treatments around potential
bleeding complications and the onset of HIT as a side effect; both of which are associated
with high dose and prolonged use. Consequently, alternative agents, which maintain
desirable antiviral properties, while offering reduced anticoagulant and HIT risk,
are sought.
PPS, which is already in use for the relief of bladder pain and discomfort associated
with interstitial cystitis, has been shown here to possess antiviral properties comparable
to those of UFH and stronger than those of LMWHs. In contrast to heparin, the lower
anticoagulant (aPTT) potency of PPS (10-fold lower compared with heparin) and lack
of anti-Xa activity could allow its administration at higher doses thereby enabling
its antiviral properties to be exploited.[38]
[39] The oligosaccharide fractions of PPS maintain the antiviral properties of the parent
intact polysaccharide and this may provide a way of avoiding potential side effects
analogous to HIT, which depend on the formation of immunogenic complexes between platelet
factor 4 and HS oligosaccharides of ca. 9 kDa or larger.[40]
Both PPS and its fractions bind the S1-RBD efficiently, with K
D values in the μM to nM range, with similar energy changes, and both involve interactions
that are driven, as expected, primarily by charge–charge interactions ([Table 3]). It is not known whether the detailed interactions of PPS and its fractions with
the protein are identical to those of heparin, but docking experiments suggest that
the interactions of both heparin and PPS with S1-RBD occur between the sulfate groups
of the xylohexasaccharide and the site I sequence of S1-RBD (N354, R355, K356, R357).[6]
[7] An interesting observation is the change from the normally predominant 4C1 chair form of unsulfated pentosan and xylans[41] to the 1C4 conformation for the polysulfated xylose residues. This is presumably the result
of repulsion between the charged sulfate groups in positions 2 and 3, favoring the
lower energy 1C4 conformation and a greater distance between the two sulfate groups ([Supplementary Fig. S5], available in the online version).
The far-UV CD spectra of the S1-RBD alone and the protein in the presence of PPS,
while closely similar, are, nevertheless, not identical ([Table 4]). While this is broadly consistent with there being no major change of secondary
structure, minor changes in the protein secondary structure cannot be excluded. The
origin of such differences may reside in subtle distinction between the modes of binding
of these two polyanions.
It has been noted that, owing largely to our lack of precise understanding at the
molecular level of many viral invasion mechanisms, it is probable that most current
antiviral strategies are not optimised[42] and this is certainly true for the inhibition of invasion by SARS-CoV-2. It is also
noteworthy that both SARS-CoV (CoV-1) and the present SARS CoV-2 have been found to
be susceptible to inhibition by anionic polysaccharides,[6]
[8] and this class of compounds warrants more extensive investigation for the inhibition
of viral invasion. Furthermore, PPS and fractions derived from it exhibit reduced
anticoagulant activity for the inhibition of invasion of susceptible cells by SARS
CoV-2 and merit further investigation for potential use as a treatment. While maintaining
a better level of inhibitory potential on a weight per volume basis than LMWH and
similar to that of UFH, PPS exhibits lower anticoagulant activity. Therefore, PPS
should be less prone to bleeding complications at high doses and, by virtue of its
distinct structure and the maintenance of antiviral activity in fractions derived
from it, offers an alternative that is likely to be free of HIT or analogous side
effects. Concentrations of PPS effective for antiviral activity can be reached at
the level of the luminal surface of the first respiratory tract by using nebulization
as a route of administration. This strategy has already been tested for heparin.[43] The starting material is derived from pentosan, a plant-derived polysaccharide xylan
with a well-defined repeating structure[19] and, even following a chemical sulfation step, it remains highly homogeneous, making
it easier to ensure both consistency and to avoid contamination, two problems that
have dogged heparin production in recent years.[44]
[45]
What is known about this topic?
-
The pandemic COVID-19 disease is of global significance and an international health
emergency.
-
Additional treatments to vaccines are required.
-
Heparin inhibits cellular invasion of SARS-CoV-2 coronavirus in live virus assays.
-
Heparin efficacy appears to depend largely in its interactions with the spike protein
of SARS-CoV-2.
-
The occurrence of bleeding complications and the documented risk of heparin-induced
thrombocytopenia (HIT) after prolonged exposure both limit the use of heparin in patients
having high incidence of VTE, particularly for high-dose administration.
-
Several studies have suggested that other sulfated molecules and highly charged polyelectrolytes
also inhibit cellular invasion of SARS-CoV-2 coronavirus in live virus assays.
-
Pentosan polysulfate, a plant-derived xylan that has been approved for the treatment
of bladder pain and discomfort in interstitial cystitis, could represent an alternative
to heparin.
What does this paper add?
-
Pentosan polysulfate has been shown to possess antiviral properties comparable to
those of UFH and stronger than those of LMWH.
-
The lower anticoagulant potency of PPS and lack of anti-Xa activity could allow its
administration at higher doses thereby enabling its antiviral properties to be exploited.
-
Both PPS and its fractions bind the S1-RBD efficiently, with K
D values in the μM to nM range,
-
PPS polysulfated xylose residues change their conformation from the normally predominant
4C1 chair form of unsulfated pentosan and xylans[39] to the 1C4 conformation.
-
Like heparin, the interaction of PPS with S1-RBD occurs between the sulfate groups
of the xylohexasaccharide and the site I sequence of S1-RBD.
-
The starting material is derived from a plant-derived polysaccharide xylan with a
well-defined repeating structure and, even following a chemical sulfation step, it
remains highly homogeneous, making it easier to ensure both consistency and to avoid
contamination.
