Article
Reference
Modified cyclodextrins as broad-spectrum antivirals
JONES, Samuel T, et al.
Abstract
Viral infections kill millions of people and new antivirals are needed. Nontoxic drugs that irreversibly inhibit viruses (virucidal) are postulated to be ideal. Unfortunately, all virucidal molecules described to date are cytotoxic. We recently developed nontoxic, broad-spectrum virucidal gold nanoparticles. Here, we develop further the concept and describe cyclodextrins, modified with mercaptoundecane sulfonic acids, to mimic heparan sulfates and to provide the key nontoxic virucidal action. We show that the resulting macromolecules are broad-spectrum, biocompatible, and virucidal at micromolar concentrations in vitro against many viruses [including herpes simplex virus (HSV), respiratory syncytial virus (RSV), dengue virus, and Zika virus]. They are effective ex vivo against both laboratory and clinical strains of RSV and HSV-2 in respiratory and vaginal tissue culture models, respectively. Additionally, they are effective when administrated in mice before intravaginal HSV-2 inoculation. Lastly, they pass a mutation resistance test that the currently available anti-HSV drug (acyclovir) fails.
JONES, Samuel T, et al. Modified cyclodextrins as broad-spectrum antivirals. Science Advances, 2020, vol. 6, no. 5, p. eaax9318
DOI : 10.1126/sciadv.aax9318 PMID : 32064341
Available at:
http://archive-ouverte.unige.ch/unige:139761
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advances.sciencemag.org/cgi/content/full/6/5/eaax9318/DC1
Supplementary Materials for
Modified cyclodextrins as broad-spectrum antivirals
Samuel T. Jones, Valeria Cagno, Matej Janeček, Daniel Ortiz, Natalia Gasilova, Jocelyne Piret, Matteo Gasbarri, David A. Constant, Yanxiao Han, Lela Vuković, Petr Král, Laurent Kaiser, Song Huang, Samuel Constant, Karla Kirkegaard,
Guy Boivin, Francesco Stellacci*, Caroline Tapparel*
*Corresponding author. Email: caroline.tapparel@unige.ch (C.T.); francesco.stellacci@epfl.ch (F.S.) Published 29 January 2020, Sci. Adv. 6, eaax9318 (2020)
DOI: 10.1126/sciadv.aax9318
This PDF file includes:
Section S1. Synthesis, characterization, and assays
Fig. S1. Reaction scheme overview of the synthesized modified CDs.
Fig. S2. Characterization of CD1.
Fig. S3. Characterization of CD2.
Fig. S4. Characterization of CD3.
Fig. S5. Cholesterol replenishment.
Fig. S6. Viral yield reduction assay of DENV-2.
Fig. S7. Virucidal activity and ex vivo inhibition against ZIKV.
Fig. S8. Viral inhibition in the presence or absence of serum.
Fig. S9. Simulations of CDs with gB fusion loops.
Fig. S10. Toxicity of CD1 in respiratory tissues.
References (35–42)
Supplementary Materials References: 35-42.
S.1.1 Mass Spectroscopy.
Mass spectrometry analyses were performed on a LTQ Orbitrap Elite FTMS instrument (Thermo Scientific, Bremen, Germany) operated in the negative mode coupled to a modified HESI-II probe in an Ion Max ion Source able to perform Cold-spray ionization (CSI). Cold-spray ion- ization is an ESI-MS modification operated at very low temperature that can be used to prevent decomposition of labile molecules that are difficult to observe by conventional MS techniques.
The experimental conditions for the ionization voltage were -1.2kV. The temperature of ion transfer capillary was 60◦C, tube voltages. Sheath and auxiliary gases were 35 and 10 respec- tively to obtain a spray temperature around +5◦C. The infusion rate was 5 µ
spectra were obtained in the 80-1000 m/z range in the reduce profile mode with a resolution set to 120,000 and automatic gain control (AGC) value set at 10+6. A total of 100 scans each consisting in 10µscans were acquired in reduced profile mode and averaged.
S.1.2 CE-UV.
Synthesised compounds were analysed by capillary electrophoresis (CE) with UV detection (CE-UV) following the protocol described by the United States Pharmacopeia (USP) and the National Formulary (NF) with minor modifications (Betadex Sulfobutyl Ether Sodium USP NF 33, Official Monograph, Betadex 6546-6550). CE-UV experiments were carried out using a 7100 A CE apparatus (Agilent, Waldbronn, Germany) and untreated fused silica separation capillary (BGB analytik AG, Böckten, Switzerland) of 50µm i.d., 40.5 and 49 cm of effective and total length, respectively. Background electrolyte (BGE) was 30 mM benzoic acid with pH 8 adjusted by addition of 100 mM Tris solution. Prior to sample analysis each new capillary was conditioned by flushing for 10 min with 1 M NaOH, followed by 5 min flushing with 0.1 M NaOH and then 5 min with milliQ H2O. CE-UV analysis program consisted of the following steps: a) flushing with 0.1 M NaOH for 2 min; b) flushing with milliQ H2O for 2 min; c) flushing with BGE for 2 min; d) sample injection for 5 sec with 40 mbar pressure; e) application of 30 kV separation voltage for 10 min. UV signal was monitored at 230.0 nm wavelength. Peak assignment was performed with captisol as a reference compound taking into account capillary- to-capillary migration time variation.
S.1.3 Molecular dynamics simulations.
Atomistic molecular dynamics (MD) simulations were performed of cyclodextrin (CD) inter- acting with Herpes Simplex Virus (HSV) Glycoprotein B (gB). Three different βCDs were simulated (CD1, CD2andCD3). The structure of gB was based on the structure with pdb ID
Section S1. Synthesis, characterization, and assays
35
l/min. FTMS
5FZ2; each protein unit of the tetramer contained residues 142 to 476, and the missing atoms in the structure were added with the VMD37plugin psfgen.
In the simulations, 10 CD molecules were initially placed near the fusion loops of gB and solvated in a 0.15 M NaCl solution. The key amino acid residues of gB fusion loop that are responsible for the cell fusion activity are known (Trp 174, Phe 175, Gly 176, Tyr 179, Ala 261, His 263, Arg 264). Unit cells of the simulated systems, containing CDs and gB in aqueous solution, had in total between 400,000 and 500,000 atoms.
The CD ligands were described with the CHARMM general force field and proteins were de- scribed with the CHARMM36 protein force field. The simulations were performed with NAMD2.12. The particle-mesh Ewald (PME) method was used for the evaluation of long- range Coulomb interactions. The time step was set to 2.0 fs, and long-range interactions were evaluated every 1 (van der Waals) and 2 timesteps (Coulombic). After 2,000 steps of mini- mization, ions and water molecules were equilibrated for 2 ns around gB and CDs, which were restrained using harmonic forces with a spring constant of 1 kcal/(mol Å2). For all the systems, the last frames of restrained equilibration were used to start simulations of partially restrained proteins (restraints were placed on the atoms of the bottom part of the red domains in Figure 1) and free CDs. All the simulations were performed for 50 ns in the NPT ensemble (pressure p
= 1 bar and temperature T = 300 K), using the Langevin dynamics (γLang = 1 ps-1).
S.1.4 Cholesterol replenishment.
Vero cells were seeded in 24-well plates. The following day mixtures of CD1 (100 µg/ml) or methyl-beta-CD (3 mg/ml) and HSV-2 were incubated for 1h at 37 ◦C and subsequently subjected to cholesterol replenishment. 1 mM of cholesterol or the equal volume of ethanol were added for 30 minutes at 37◦C. After they were added for 2 hours at 37◦C on Vero cells.
The inocula were subsequently removed from the wells, and the cells were washed with medium twice and overlaid with a medium containing 1.2% methylcellulose. After 24 hours cells were stained and plaques were counted in order to determine % of infection.
S.1.5 Lactate dehydrogenase assay (LDH).
LDH release in basal medium was measured with the Cytotoxicity Detection Kit (Roche 04744926001).
S.1.6 ELISA.
Interleukin-6 (IL-6), CXC motif chemokine 10 (CXCL10 or IP-10), CC motif chemokine5 (CCL5 or RANTES), interleukin-8 (IL-8 or CXCL-8) and interferon lambda (IL-29 /IL-28B) were measured in the basal and apical medium by ELISA (R&D DY206-05, DY266-05, DY278- 05, DY208-05 and DY1598B-05) following everyday treatment with different concentrations of CD 1.
36
38
39,40
41 42
O HO OH O
SH
7
Na+ S
O O O-
S O
O O- NH
O
Na+
R O
HO OH O S
7 hv (UV, 250 W)
18h
Na+ S
O O O- R =
CD1 -
CD2 -
CD3 -
R
Fig. S1. Reaction scheme overview of the synthesized modified CDs.
3 4 5 6 7 8 9 420
440 460 480 500
6 7
bCD-MUS
Time / min
UV / mAU
CD1
a)
b)
c)
a) HRMS Cold-Spray Ionisation spectrum of CD1 in EtOH/H2O (50:50), b) HRMS Summary ofCD1in EtOH/H2O (50:50). Values correspond to the monoisotopic mass and c) CE-UV electropherogram ofCD1 at 230 nm with assigned CD species showing 31% of 6 and 69% of 7 sulfonates per CD.
Fig. S2. Characterization of CD1.
3 4 5 6 7 8 9 80
100 120 140
3
bCD-C7-SO3Na
UV / mAU
Time / min 5
7
CD2
!"#$%&#'()*"(+&#' ,-$"($./%'#)01'2 345$(/+$6.'#)01'2) 3(("()055+2)
7089:;<=>:?@A208A;B9@>=2ACAD =EEFG?HG =EEFG??? GFE
7089:;<=>:?@A208A;B9@>=2AI'C<D 9B?FBG:B 9B?FBG:= GFE
7089:;<=>:?@A208A;B9@>=2AI':CED EG<F=:G= EG<F=:GB GF9
7089:;<=>:?@A208A;B9@>=2<C<D =?9FEH:A =?9FEH9: GFA
7089:;<=>:?@A208A;B9@>=2ECED 9:EF?HAH 9:EF?H?: GF?
a)
b)
c)
a) HRMS Cold-Spray Ionisation spectrum of CD2 in EtOH/H2O (50:50), b) HRMS Summary ofCD2in EtOH/H2O (50:50). Values correspond to the monoisotopic mass and c) CE-UV electropherogram ofCD2 at 230 nm with assigned CD species showing 6% of 3, 32.7% of 5 and 61.3% of 7 sulfonates per CD.
Fig. S3. Characterization of CD2.
3 4 5 6 7 8 9 10 90
100 110 120 130 140
7 8
bCD-Ar-SO3Na
UV / mAU
Time / min
450 500 550 600 650 700 750 800 850 900 950 1000 0.0
2.0x104 4.0x104 6.0x104 8.0x104 1.0x105 1.2x105 1.4x105 1.6x105
Intensity
m/z
471.9668 z=8
494.0951 z=7
580.2758 z=6 542.6748 z=7
700.9272
z=5 876.1593
z=4 636.9525
z=6
768.9409 z=5
!"#$%&'()%*+,-"#.'&./0)$+-*1*2
!"#$%&#'()*"(+&#' ,-$"($./%'#)01'2 345$(/+$6.'#)01'2) 3(("()055+2)
7089:;<=>:?@A208B<;BCD@>92?E?F 9ABGH<ABB 9ABGH<<? IG<
7089:;<=>:?@A208B<;BCD@>92?D'EAF C9:G<ACB C9:G<A9? IG<
7089:;<=>:?@A208B<;BCD@>92AEAF 9H9GIH9? 9H9GIHC: IG?
7089:;<=>:?@A208B<;BCD@>92AD'E<F C?IG:ACC C?IG:AC? IGC
7089:;<=>:?@A208A;B9@>=2<E<F C:=GCH?C C:=GCH?B IG?
CD3
a)
b)
c)
a) HRMS Cold-Spray Ionisation spectrum of CD3 in EtOH/H2O (50:50), b) HRMS Summary ofCD3in EtOH/H2O (50:50). Values correspond to the monoisotopic mass and c) CE-UV electropherogram ofCD3 at 230 nm with assigned CD species. Integration was not possible due to low intensity of peaks.
Fig. S4. Characterization of CD3.
CD1 or methyl-beta-CD were incubated for 1 h at 37
◦C with HSV-2. Following this incubation 1 mM of cholesterol or the corresponding volume of ethanol were added to the samples for 30 min. The mixtures was then added on cells and
UT 300 100 33.33 11.11 3.70 1.23
100101 102 103 104 105 106 107 108
T C ID 5 0 /m l
μg/ml
CD1
****
***
BHK-21 were infected with DENV-2 at a MOI of 0.1, 2 hpi the virus was washed out and serial dilutions of CD1 were added.
Supernatants were collected 48 hpi and titrated by TCID50. Results are mean and SEM of 2 independent experiments. UT, untreated. **** p<0.0001 *** p<0.0005
Fig. S5. Cholesterol replenishment.
Fig. S6. Viral yield reduction assay of DENV-2.
CD1 100 M CD 3 mg/ml 0
50 100
%o fi nfectio n
- CHOLESTEROL+ CHOLESTEROL
μg/ml β
the infectivity evaluated through plaque assay. Results are mean and SEM of 2 independent experiments.
ZIKV ZIKV + CD1 102
103 104 105 106 107 108
TCID50/ml
0 24 48 72 96 120 144
3 4 5 6 7 8
hpi
Logrnacopies/tissue
ZIKV ZIKV + CD1
a) b)
a) Virucidal activity ofCD1 against ZIKV: ZIKV was incubated withCD1(30µg) for 1h and then serially diluted on Vero cells and b) ZIKV inhibition in vaginal tissues: Tissues were infected in presence or absence of 100µg/ml ofCD1. After 4 h the inocula were removed and tissues were washed extensively.
Every 24 h after that, 200 µl of culture medium was applied apically for 30min at 37◦C for collection of apically released viruses. RNA copies were evaluated with qPCR. Results are mean and SEM of 2 independent experiments.
-3 -2 -1 0 1 2 3
0 50 100
Logµg/mL
%ofinfection
FBS 2.5% (EC5028.9µg/ml) FBS 0% (EC500.0433µg/ml)
-1 0 1 2 3
0 50 100
Logµg/mL
%ofinfection
FBS 2.5% (EC5021.2µg/ml) FBS 0% (EC503.53µg/ml)
A B
a) HSV-2 and b) RSV-A inhibition assays were conducted in presence of 2.5% FBS as in all the other experiments presented in the paper or in presence of medium. Results are mean and SEM of 2 independent experiments.
Fig. S7. Virucidal activity and ex vivo inhibition against ZIKV.
Fig. S8. Viral inhibition in the presence or absence of serum.
serum-free
45 47 49 51 53 55 -20
-15 -10
Energy (Kcal/mol)
t (ns) CD1 CD2
CD3
a)
b)
a) Fusion loops of gB protein (circled green) with inter-loop distances (d1, d2 and d3) identified. b) Averaged binding energy of CD molecules to gB fusion loop (energy values are reported per single CD molecule; smoothing of data was performed by adjacent averaging method).
Fig. S9. Simulations of CDs with gB fusion loops.
pg/ml
IL-6 IL-29 IL-8 RANTES IP-10 100
101 102 103 104 105
2000µg/mL 1000µg/mL 500µg/mL Mock
pg/ml
IL-6 IL-29 IL-8 RANTES IP-10 100
101 102 103 104 105
2000µg/mL 1000µg/mL 500µg/mL Mock
pg/ml
IL-6 IL-29 IL-8 RANTES IP-10 100
101 102 103 104 105
2000µg/mL 1000µg/mL 500µg/mL Mock
pg/ml
IL-6 IL-29 IL-8 RANTES IP-10 100
101 102 103 104 105
2000µg/mL 1000µg/mL 500µg/mL Mock
Ω/cm3
Mock 2000 1000 500 Triton 0
200 400 600
µg/mL
%ofcytotoxicity
24hpt 48hpt 72hpt 96hpt 0
50
100 2000µg/mL
1000µg/mL 500µg/mL Triton
A B
C D
E F
a-d) Cytokine/chemokine levels by ELISA assays a) in basal medium, 2 days post a single administration of the indicated concentration of CD1 b) in basal medium four days after daily treatment with the indicated concentration of CD1 c) in apical sample collected 2 days post a single administration of the indicated concentration of CD1, d) in apica l sample s collecte d fo ur da ys af ter da ily treatm ent w ith the indic ated concentrationofCD1. e) Transepithelial electrical resistance (TEER) of tissues measured at the
f) LDH release every 24 h after treatment.
Fig. S10. Toxicity of CD1 in respiratory tissues .
indicated time post daily treatment with the indicated concentration of CD1.
