Increased in vitro Anti-HIV Activity of Caffeinium-Functionalized Polyoxometalates

Article abstract of DOI:10.1002/cmdc.202100281

Polyoxometalates (POMs), molecular metal oxide anions, are inorganic clusters with promising antiviral activity. Herein we report increased anti-HIV-1 activity of a POM when electrostatically combined with organic counter-cations. To this end, Keggin-type cerium tungstate POMs have been combined with organic methyl-caffeinium (Caf) cations, and their cytotoxicity, antiviral activity and mode of action have been studied. The novel compound, Caf₄K[β₂-CeSiW₁₁O₃₉]×H₂O, exhibits sub-nanomolar antiviral activity and inhibits HIV-1 infectivity by acting on an early step of the viral infection cycle. This work demonstrates that combination of POM anions and organic bioactive cations can be a powerful new strategy to increase antiviral activity of these inorganic compounds.

Full text of DOI:10.1002/cmdc.202100281

Accepted Article
Title: Increased in-vitro Anti-HIV Activity of Caffeinium-functionalized
Polyoxometalates
Authors: Ana G. Enderle, Matteo Bosso, Rüdiger Groß, Magdalena
Heiland, Mariela Bollini, María J. Culzoni, Jan Münch, and
Carsten Streb
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To be cited as: ChemMedChem 10.1002/cmdc.202100281
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1/2020

ChemMedChem
10.1002/cmdc.202100281
FULL PAPER
Increased in-vitro Anti-HIV Activity of Caffeinium-functionalized
Polyoxometalates
Ana G. Enderle#[a,b,c], Matteo Bosso#[d], Rüdiger Groß , Magdalena Heiland , Mariela Bollini , María J.
[d]
[a]
[b]
[
c]
[d]
[a]
Culzoni , Jan Münch* and Carsten Streb*
[a]
[b]
[c]
[d]
Dr. A. G. Enderle, Prof. Dr. C. Streb
Institute of Inorganic Chemistry I
Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
E-mail: Carsten.streb@uni-ulm.de
Dr. A. G. Enderle, Dr. M. Bollini
Medicinal Chemistry Lab
Centro de Investigaciones en Bionanociencias (CIBION), CONICET
Godoy Cruz, 2390, Ciudad de Buenos Aires, Argentina
Dr. A. G. Enderle, Prof, Dr. M. J. Culzoni
Laboratorio de Desarrollo Analítico y Quimiometría (LADAQ)
Universidad Nacional del Litoral - CONICET
Ciudad Universitaria, Paraje El Pozo, CC242, S3000, Santa Fe, Argentina
Dr. M. Bosso, M.Sc. R. Groß, Prof. Dr. Frank Kirchhoff, Prof. Dr. J. Münch
Institute of Molecular Virology
Ulm University Medical Center
Meyerhofstraße 1, 89081 Ulm, Germany
E-mail: jan.muench@uni-ulm.de
#
These authors contributed equally
Supporting information for this article is given via a link at the end of the document.
Abstract: Polyoxometalates (POMs), molecular metal oxide anions,
simplest and most versatile strategy is the electrostatic
combination of POM anions with organic cations, leading to the
formation of organic-inorganic POM salts which combine the
properties of both components.[ This principle has for example
been used to combine POM anions with bactericidal tetra-
alkylammonium cations to give POM composites with high
are inorganic clusters with promising antiviral activity. Here, we report
increased anti-HIV-1 activity of
a POM when electrostatically
13]
combined with organic counter-cations. To this end, Keggin-type
cerium-tungstate POMs have been combined with organic methyl-
caffeinium (Caf) cations, and their cytotoxicity, antiviral activity and
mode of action have been studied. The novel compound, Caf
4
K[β
2
-
antibacterial efficiency. [14–16]
.
CeSiW11 O, exhibits sub-nanomolar antiviral activity and
O
39]×H
2
Here, we build on these concepts and explore how cerium-
functionalized tungstate Keggin POMs can be combined with
organo-cations, to improve antiviral activity against HIV-1
infection. As a model system, we selected the caffeinium cation,
because the precursor, caffeine, is a natural compound with
various promising therapeutic applications and expected to be
non-toxic.[17] In addition, earlier studies suggested that caffeine
and caffeine-related methylxanthines can affect HIV replication,
inhibits HIV-1 infectivity by acting on an early step of the viral infection
cycle. This work demonstrates that combination of POM anions and
organic bioactive cations can be a powerful new strategy to increase
antiviral activity of these inorganic compounds.
Introduction
[18,19]
by acting on the integration step of the virus life cycle.
In this project, we report caffeinium-functionalized POM species
and provide initial insights into their anti-HIV activity together with
mechanistic understanding of the combined mode of action of the
cation and anion moieties. This new approach could open the
pathway for bringing together organic and inorganic antiviral
agents to harness their full synergistic potential in developing
next-generation antiviral treatments.
One intriguing class of inorganic materials which has attracted
significant interest in the field of medicinal chemistry are
molecular metal oxides, or polyoxometalates (POMs). POMs are
metal-oxo anions based on high-valent transition metals, often Mo
or W.[1] Pioneering studies have shown that POMs are promising
materials with antibacterial, antiviral and antitumor properties.[2–5]
One typical approach to tune POM reactivity is their
functionalization by incorporation of hetero-elements such as
metal cations. This concept has been successfully employed to
design POMs with antiviral properties, and a range of metal-
functionalized POMs have been reported as broad-spectrum
inhibitors of enveloped DNA and RNA viruses as well as
retroviruses such as HIV-1 and HIV-2.[6–10] In one study, it was
shown that the sandwich-type cerium-functionalized species
Results and Discussion
Briefly, the caffeinium-containing POM-salt was synthesized by
cation metathesis as follows: the mono-cerium-functionalized
lacunary Keggin anion was obtained by reaction of Ce(III) with the
monolacunary K
CeSiW11
and analytical details see SI). The methyl-caffeinium cation
,3,7,9-tetramethyxanthinium (hereafter: CAF) was synthesized
by methylation of caffeine (for details see SI). The caffeinium-
8
[β
2
-SiW11
O39] × 13H
2
O in water to give K
5 2
[β -
39 2 2
K13[Ce(SiW11O ) ] × 26H O is one of the most potent anti-HIV
O
39] × H
2
O (hereafter: K
5
Ce-POM × H
2
O, for synthetic
POMs, with an IC50 of ~ 30 nM by binding to the viral envelope
protein gp120.[11] Their functionalization with covalently or
noncovalently linked organic species is an additional approach to
introduce secondary bioactivity into POMs.[12] Arguably the
1
1
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ChemMedChem
10.1002/cmdc.202100281
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measuring intracellular ATP levels. Values shown are mean values derived from
one experiment performed in triplicates (± standard deviation).
4 2 2
POM compound Caf K[β -CeSiW11O39]×H O (Caf-POM) was
obtained by combining aqueous solutions of CAF and Ce-POM,
and selective precipitation of the product. Sample identification
and purity confirmation were performed by NMR spectroscopy,
CHN elemental analysis, inductively coupled plasma optical
emission spectroscopy (ICP-OES) and FT-IR spectroscopy (for
details see SI). The stability of the compound under the
experimental conditions in aqueous phosphate buffer solution
To verify that the observed antiviral activity is not due to a
potential cytopathic/cytotoxic effect on TZM-bl cells, a cell viability
assay was performed. None of the compounds reduced cellular
metabolic activity when tested at final cell culture concentrations
of up to 100 μM, which are orders of magnitude higher than the
antiviral activities (Fig. 2).
Previous studies suggested that POMs inhibit virion attachment
to the target cell [14]. To assess whether Caf-POM act by a similar
mechanism, HIV-1 pseudoparticles were first exposed to the
compound for 15 min and then used to inoculate TZM-bl cells.
Alternatively, cells were treated with Caf-POM for 24 hours,
washed to remove unbound compound and then infected.
Infection rates were determined two days later and confirmed a
sub-nanomolar IC50 of 0.9 nM of Caf-POM under the conditions of
virion treatment (Fig. 3). In contrast, Ca-POM lost a considerable
amount of its antiviral activity if removed from the cells prior to
infection. This finding suggests that the Ca-POM targets the viral
particle and/or prevents its interaction with the target cell.
(
PBS) at pH 7.4 (10 mM) was analyzed by UV-Vis spectroscopy
and showed no significant changes over the course of one week
for details see SI).
(
Figure 1. Effect of Caf-POM und Ce-POM on a) HIV-1 gp120/41 and b) VSV-
G pseudotype infection. Virions were exposed to serial dilutions of the
compounds and used to inoculate TZM-bl cells. 2 days later, infection rates were
determined by quantifying ß-galactosidase activities in cellular lysates. Shown
are mean values derived from three independent experiments each performed
in triplicates (± standard deviation).
The antiviral activity of Caf-POM and Ce-POM (reference
compound) was first evaluated against lentiviral pseudotypes
carrying either the glycoproteins of HIV-1 (HIV-1 gp120/41), or the
vesicular stomatitis virus (VSV-G; for details see SI). For this,
virions were treated with the compounds and then used for
inoculation of TZM-bl reporter cells. Infection rates were
determined 2 days later by a luminescence based assay (see SI)
and revealed a concentration-dependent inhibition of both viral
pseudotypes (Fig. 1) All HIV-1 stocks were prepared in DMEM
medium supplemented with 10 % fetal calf serum. Both POMs
inhibited HIV-1 pseudotype infection, with Caf-POM being ~15-
fold more potent (IC50: 0.3 nM) than Ce-POM (IC50: 4.6 nM) (Fig.
Figure 3. Caf-POM inhibits an early step in the HIV-1 life cycle. Caf-POM
was either pre-incubated with TZM-bl reporter cells (blue lines) for one day prior
to infection, or mixed with HIV-1 pseudoparticle (black lines) and then added
onto TZM-bl cells. Infection rates were determined two day later by quantifying
ß-galactosidase activities in cellular lysates. Shown are mean values derived
from three independent experiments each performed in triplicates (± standard
deviation).
1
a). For VSV-G pseudotypes, antiviral efficacy was generally
lower, with IC50 values of Ce-POM of 13.7 nM and Caf-POM of
11 nM. Thus, Caf-POM exerts a high sub-nanomolar activity
1
Having demonstrated that the Caf-POM may target an early step
in the viral life cycle, we next investigated whether the time the
virions are exposed to the POMs affects antiviral activity. HIV-1
pseudoparticles were incubated for 360, 180, 60, 30, 15 or 0 min
with both POMs, and then the mixtures were used to infect TZM-
bl cells. Infection rates were determined two days later and
showed a time-dependent increase in the antiviral activity of the
Caf-POM (Fig. 4a, 4c) and the Ce-POM (Fig. 4b, 4c). For example,
an IC50 value of 483 pM was obtained when the Caf-POM was
mixed with HIV-1 and immediately added to the cells (0 min) (Fig.
against HIV-1.
4a, 4c). After 1 h of incubation, the IC50 decreased to 104 pM, and
after 6 hours to only 53 pM (Fig. 4a, 4c), corresponding to a 9-fold
enhanced antiviral effect. Ce-POM (IC50: 4.950 pM) inhibited
infection at the 0 min time point ~ 10-fold less effectively as Caf-
POM (IC50 483 pM), largely confirming observations shown in Fig.
1. However, with increasing incubation times, a comparably more
pronounced antiviral effect was observed for the Ce-POM, with
an IC50 value of only 96 pM at 360 min, corresponding to more
than 50-fold enhanced antiviral effect. Collectively, these findings
Figure 2. Effect of Caf- and Ce-POMs on the metabolic activity of TZM-bl
cells. 10.000 TZM-bl reporter cells were incubated for 48 hours with the
indicated concentrations of POMs before cell viability was determined by
2
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ChemMedChem
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suggest that both POMs target the viral particle in a time
dependent manner. The Caf-POM shows faster viral inactivation
kinetics as the Ce-POM, but both compounds exert almost similar
antiviral activities if incubated with the HIV-1 particles for 6 hours.
It is suggested that beyond the electrostatic interaction of the
components of Caf-POM, there could be an additional face-to-
face stacking (lone pair-π hole interaction) between the purine
base and the Ce-POM. It was reported that these interactions
occur at the largest faces of the anion.[21] In this structure, this
+
+
could be the reason for the presence of one K and four Caf to
balance the charges as the POM has four largest faces (leaving
the large faces nearby the cerium discriminated for steric
hindrance effect). POMs can interact with different amino acids of
viral proteins, mainly through electrostatic interactions and
hydrogen bonds. In addition to this, the caffeinium compound
could introduce cooperative effects through the inclusion of a
second noncovalent interaction (i.e. π-stacking or cation–π
interactions) to form anion–π–π and anion–π–cation triads with
the amino acids situated at the protein surface.[22] All these
interactions could play an important role in the mechanism of
action of Caf-POM against the HIV infection.
Figure 4. The antiviral activity of Caf- and Ce-POMs increases over time.
HIV-1 particles were exposed for indicated times to serial dilutions of Caf-POM
(a) and Ce-POM (b). Thereafter, mixtures were used to inoculated on TZM-bl
cells and infection rates were determined two days later. Shown are mean
values ± standard deviation of two independent experiments performed in
triplicates. c) IC50 values of experiments shown in a and b were calculated using
Graph Pad Prism. Statistical differences between IC50 values was assessed with
the unpaired t test. * p < 0.05; ** p < 0.01; n = 2 ± SEM.
To gain more insight into the antiviral mechanism, a liposome
leakage assay was performed which uses virion-mimicking lipid
vesicles to evaluate membrane-disrupting activity. This is an
important criterion, as envelope disruption would irreversibly
inactivate HIV-1 virions and could also indicate broad antiviral
activity. To this end, vesicles containing self-quenching
concentrations of carboxyfluorescein dye were treated with Caf-
POM or Ce-POM. Leakage of the fluorescent dye and thus
disruption of the vesicle membrane was then assessed by
emission spectroscopic analysis of the reaction solution over 60
min. Here, we utilized vesicles consisting of DOPC/SM/Chol (for
details see SI, molar ratio 45:25:30 mol-%) to mimic the HIV viral
membrane. Interestingly, both POM exerted dose-dependent
membrane-disrupting activity. A substantially higher and more
rapid effect was observed for Caf-POM as compared to Ce-POM
(
Fig. 5a), highlighting that the combined action of cation and anion
results in an accelerated membrane-disruptive action. Of note,
DMSO only did not induce liposome leakage (Fig. 5b) and POMs
itself did not exhibit autofluorescence (Fig. 5c) demonstrating that
the observed changes in fluorescence are caused by released
carboxyfluorescein. The disruption of the lipid membrane could
be explained by a hydrolytic mechanism based on the Lewis-
acidic cerium(III) ion in the POM. [20] However, the concentrations
of both POMs required for substantial membrane-disrupting
activity within 1h are in the micromolar range, indicating that (also)
other mechanisms are likely involved in antiviral activity observed
at picomolar concentrations.
Figure 5. POMs disrupt membranes of virus-like liposomes. Virus-like
liposome (VLL) with a membrane composition of DOPC/SM/Chol (45/25/30
3
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ChemMedChem
10.1002/cmdc.202100281
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mol%) were synthesized with 50 mM carboxyfluorescein cargo and purified by
2
980–2999.
9
SEC. 2.25
x
10 particles were added to 96 well plates and baseline
[
[
6]
7]
M. Witvrouw, H. Weigold, C. Pannecouque, D. Schols, E. De
Clercq, G. Holan, J. Med. Chem. 2000, 43, 778–783.
X. Wang, J. Wang, W. Zhang, B. Li, Y. Zhu, Q. Hu, Y. Yang, X.
Zhang, H. Yan, Y. Zeng, Viruses 2018, 10, 265.
fluorescence measured for 5 min while incubating at 37 deg C. (a) Indicated
concentrations of POMs were then added (first dashed line) and plates
incubated for 1h. Finally, Triton X-100 was added to all wells at a final
concentration of 1% (second dashed line) to induce total lysis; values were
baseline-subtracted and normalized to the signal obtained for total lysis. (b)
DMSO equivalent to the amounts used in POM preparations did not induce
leakage. (c) POM in PBS at the highest dose tested (100 μΜ) did not exhibit
autofluorescence or an increase in fluorescence after 1h incubation. Data
shown are means ± SEM form two experiments performed in triplicates.
[8]
J. Wang, Y. Liu, K. Xu, Y. Qi, J. Zhong, K. Zhang, J. Li, E. Wang, Z.
Wu, Z. Kang, ACS Appl. Mater. Interfaces 2014, 6, 9785–9789.
M. Fukuma, Y. Seto, T. Yamase, Antiviral Res. 1991, 16, 327–39.
S. Shigeta, S. Mori, T. Yamase, N. Yamamoto, N. Yamamoto,
Biomed. Pharmacother. 2006, 60, 211–9.
[
[
9]
10]
[
11]
N. Yamamoto, D. Schols, E. De Clercq, Z. Debyser, R. Pauwels, J.
Balzarini, H. Nakashima, M. Baba, M. Hosoya, R. Snoeck, Mol.
Pharmacol. 1992, 42, 1109–17.
Conclusion
In conclusion, we report the design of synergistic
polyoxometalate-organocation compounds where caffeinium
cations are combined with cerium-tungstate polyoxometalates,
leading to novel, combined anti-HIV activity with negligible
cytotoxicity. The combination of organocation and POM anion
results in enhanced anti-HIV activity as shown by the significantly
lower IC50 values observed for the caffeinium-containing
compound compared with the caffeinium-free reference. In
agreement with previous POM-virus studies, the compound is
suggested to target an early step in the viral life cycle, probably
by a direct interaction with the virus resulting in reduced entry. In
addition, we observe that the compound shows modest time-
dependent increase of the antiviral activity which we propose
could be associated with the gradual destruction of the viral
particles. To gain insight into this hypothesis, a liposome leakage
assay was performed with virion-mimicking model membranes.
The results show some membrane-disrupting activity of both
POMs, but a slightly increased activity for Caf-POM. While the
effects are much lower than required to fully explain the antiviral
activity, it indicates that the POM may interact with viral
membranes, possibly leading to interference with viral attachment
and, to a lesser extent, membrane disruption and, consequently,
inactivation of the virion. In conclusion, Caf-POM represents a
highly potent anti-HIV-1 agent and its further investigation as
microbicide to block sexual e.g. HIV-1 transmission is warranted.
[12]
[13]
H. K. Daima, P. R. Selvakannan, R. Shukla, S. K. Bhargava, V.
Bansal, PLoS One 2013, 8, 1–14.
A. Misra, K. Kozma, C. Streb, M. Nyman, Angew. Chem. Int. Ed.
2020, 59, 596–612.
[
[
[
[
14]
15]
16]
17]
A. Misra, I. Franco Castillo, D. P. Müller, C. González, S.
Eyssautier-Chuine, A. Ziegler, J. M. de la Fuente, S. G. Mitchell, C.
Streb, Angew. Chem. Int. Ed. 2018, 57, 14948.
A. Misra, C. Zambrzycki, G. Kloker, A. Kotyrba, M. H. Anjass, I.
Franco Castillo, S. G. Mitchell, R. Güttel, C. Streb, Angew. Chem.
Int. Ed. 2020, 59, 1601–1605.
A.-L. Kubo, L. Kremer, S. Herrmann, S. G. Mitchell, O. M.
Bondarenko, A. Kahru, C. Streb, S. G. Mitchell, O. M. Bondarenko,
Chempluschem 2017, 82, 867–871.
N. Singh, A. K. Shreshtha, M. S. Thakur, S. Patra, Heliyon 2018, 4,
e00829.
[18]
[19]
R. Daniel, E. Marusich, E. Argyris, R. Y. Zhao, A. M. Skalka, R. J.
Pomerantz, J. Virol. 2005, 79, 2058 LP – 2065.
G. Nunnari, E. Argyris, J. Fang, K. E. Mehlman, R. J. Pomerantz, R.
Daniel, Virology 2005, 335, 177–184.
[
[
[
20]
21]
22]
M. Ghose, S. Banerjee, S. Patra, K. K. Mukherjea, J. Lumin. 2016,
180, 224–233.
O. S. Panteleieva, A. V Shtemenko, K. V Domasevitch, Inorg.
Chem. Commun. 2018, 94, 119–122.
A. Bijelic, A. Rompel, Coord. Chem. Rev. 2015, 299, 22–38.
Acknowledgements
C.S. gratefully acknowledges financial support by Ulm University
and the Deutsche Forschungsgemeinschaft DFG. M.B. and M.J.C.
gratefully acknowledge support by Agencia Nacional de
Promoción Científica y Tecnológica, Argentina (PICT 2017-3767
and PICT 2017-0340). J.M. acknowledges funding by the DFG
CRC 1279.
Keywords: Polyoxometalate • Self-Assembly • Antiviral Activity •
HIV • Cation
[
[
1]
2]
L. Cronin, A. Müller, (guest eds.), Chem. Soc. Rev. 2012, 41, 7325–
648.
J. T. Rhule, C. L. Hill, D. a. Judd, R. F. Schinazi, Chem. Rev. 1998,
8, 327–358.
7
9
[
[
3]
4]
Hasenknopf, Bernold, Front. Biosci. 2005, 10, 275.
M. B. Čolović, M. Lacković, J. Lalatović, A. S. Mougharbel, U. Kortz,
D. Z. Krstić, Curr. Med. Chem. 2020, 27, 362–379.
[
5]
A. Bijelic, M. Aureliano, A. Rompel, Angew. Chem. Int. Ed. 2019, 58,
4
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Entry for the Table of Contents
Cerium-tungstate polyoxoanions are combined with caffeninium cations to give a hybrid organic-inorganic material with anti-HIV
activity. Biochemical assays highlight the low cytotoxicity of the compounds together with their promising antiviral activity. Initial
mechanistic studies provide information on the mode of action of the compound.
Institute and/or researcher Twitter usernames: @carsten_streb; @uni-ulm;
5
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