Prodrug Activation by a Viral Protease: Evaluating Combretastatin Peptide Hybrids to Selectively Target Infected Cells

Article abstract of DOI:10.1021/acsmedchemlett.9b00058

Infections with flaviviruses such as dengue virus (DENV) are prevalent throughout tropical regions worldwide. Replication of these viruses depends on tubulin, a host cell factor that can be targeted to obtain broad-spectrum antiviral agents. Targeting of tubulin does, however, require specific measures to avoid toxic side-effects. Herein, we report the synthesis and biological evaluation of combretastatin peptide hybrids that incorporate the cleavage site of the DENV protease to allow activation of the tubulin ligand within infected cells. The prodrug candidates have no effect on tubulin polymerization in vitro and are 20-2000-fold less toxic than combretastatin A-4. Several of the prodrug candidates were cleaved by the DENV protease in vitro with similar efficiency as the natural viral substrates. Selected compounds were studied in DENV and Zika virus replication assays and had antiviral activity at subcytotoxic concentrations.

Full text of DOI:10.1021/acsmedchemlett.9b00058

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Letter
Prodrug Activation by a Viral Protease: Evaluating Combretastatin
Peptide-Hybrids to Selectively Target Infected Cells
Michael Richter, Mila M. Leuthold, Dominik K Graf, Ralf Bartenschlager, and Christian D. Klein
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00058 • Publication Date (Web): 18 Jul 2019
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Prodrug Activation by a Viral Protease: Evaluating Combretastatin
Peptide-Hybrids to Selectively Target Infected Cells
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Michael Richter[a], Mila M. Leuthold[a], Dominik Graf[a], Ralf Bartenschlager[b, c], Christian D. Klein[a, c]*
[a]
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120
Heidelberg, Germany
[b]
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, D-
69120 Heidelberg, Germany
[c]
German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany
Keywords: Dengue virus, Zika virus, Tubulin, Combretastatin, Serine protease
ABSTRACT: Infections with flaviviruses such as dengue virus (DENV) are prevalent throughout tropical regions worldwide.
Replication of these viruses depends on tubulin, a host cell factor that can be targeted to obtain broad-spectrum antiviral agents.
Targeting of tubulin does, however, require specific measures to avoid toxic side-effects. Herein we report the synthesis and biological
evaluation of combretastatin peptide-hybrids that incorporate the cleavage site of the DENV protease to allow activation of the tubulin
ligand within infected cells. The prodrug candidates have no effect on tubulin polymerization in vitro and are 20–2000-fold less toxic
than combretastatin A-4. Several of the prodrug candidates were cleaved by the DENV protease in vitro with similar efficiency as
the natural viral substrates. Selected compounds were studied in DENV and Zika virus replication assays and had antiviral activity at
subcytotoxic concentrations.
With an estimated number of over 100 million infections per
year, dengue is considered the most common vector borne virus
infection.1 The dengue virus (DENV), a distinct member of the
genus Flavivirus, is transmitted by mosquitoes of the genus
Aedes, which are widespread in tropical and subtropical regions
around the globe. While in many cases the infection is
excruciating but heals without severe consequences, it can also
develop into the potentially fatal dengue hemorrhagic fever and
shock syndrome. The average annual number of deaths is
estimated to be in the five-digit range.2,3 Thus far, no specific
antiviral treatment is available, making symptomatic therapy
and vector control the only countermeasures. The development
of dengue vaccines is highly challenging, on the one hand due
to the occurrence of four different serotypes (DENV-1 to
DENV-4) and on the other hand due to the mechanism of
antibody-dependent enhancement.4 For the only approved
vaccine (Dengvaxia®), the WHO noted an increased risk of
severe dengue for vaccinated individuals who get infected for
the first time after vaccination.5 Therefore, developing new
antiviral agents and exploring new paths in treatment remains
an important challenge.
development because they do not experience an evolutionary
pressure by antiviral treatment. Furthermore, host targets have
the potential to serve as target for broad-spectrum antiviral
drugs, which may cover, for example, all four DENV serotypes
and other flaviviruses like Zika virus (ZIKV) and West Nile
virus.7
One potential host target for the development of novel
antivirals is tubulin. Several reports indicate that microtubules
are involved in flaviviral replication and that inhibitors of
tubulin polymerization have antiviral activity. The cytoskeleton
is used by flaviviruses for intracellular transport and virus
egress.8 Acosta et al. showed a significant reduction in virus
yield caused by inhibitors of microtubule polymerization and
suggested that the structural integrity of the microtubule
network is a requirement for efficient virus entry.9 In addition,
recent work shows a crucial role of microtubules for the
replication of ZIKV.10 However, targeting host factors also
comes at the cost of side effects and potential systemic toxicity.
This is particularly true for tubulin, a prominent anti-cancer
drug target, whose ligands frequently possess considerable
systemic toxicity. Therefore, a therapeutic or preventive
exposure to tubulin ligands in the context of viral infections can
only be considered if a mechanism is in place that prevents
unspecific toxicity. We already reported prodrugs based on
combretastatin and colchicine that were activated by human
Besides targeting the viral factors required for productive
replication or involved in pathogenesis, host factors are
promising and valuable targets to combat viral infections.6 In
particular, host factors bear a lower risk of resistance
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carboxylesterase 1, an enzyme highly abundant in monocytes
and hepatocytes, the primary target cells of DENV.11 In the
present work, we report the concept of tubulin-ligand prodrugs
that are specifically activated by the viral protease.
Page 2 of 8
moieties.20 Different functional groups on both aromatic rings
can be employed, resulting in numerous structural variations
and well-defined structure-activity relationships.21–23 A major
drawback of CA4 and its analogs is their low aqueous
solubility, resulting in difficulties to develop pharmaceutical
formulations for these drugs.
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The selective activation of tubulin-ligand prodrugs requires a
mechanism, such as enzymatic cleavage, that is exclusively
present in cells infected with flaviviruses. The flaviviral RNA
genome encodes a polyprotein, which is processed by the viral
protease NS2B-NS3.12 In the present study, we evaluated the
capability of flaviviral proteases to hydrolyze and thereby
activate peptide-hybrids of the tubulin ligand combretastatin.
Some examples have already been described in which drugs are
activated or released by a similar mechanism. Yasobu et al.
reported a colchicine prodrug that is released upon hydrolysis
by cathepsin B.13 In the field of tumor research, several
antitumor drugs are activated by proteases which are
overexpressed in tumor cells.14
Fig. 219 shows the essential structural elements of CA4
analogs that are required for activity: These are the methoxy
groups and the cis-configured double bond. Not essential is the
hydroxyl group at position B-3, which can therefore be
modified to attach the protease recognition sequence. Two
widely known examples for this approach are the phosphoric
acid ester prodrug fosbretabulin24 – developed with the
intention to increase aqueous solubility – and the serine ester
prodrug ombrabulin.25 Fosbretabulin showed anti-tumor
activity in phase II clinical trials.26 Other modifications in the
B-3 position include the design of peptidic prodrugs, such as
poly-L-Glu derivatives, to achieve a specific uptake of CA4
into tumor cells.27 Notably, the concept of peptidic CA4
prodrugs that incorporate cationic residues does not only allow
site-specific activation by the DENV protease, but it is also
expected to modulate the physicochemical properties of the
molecules and increase their solubility by introduction of a
highly polar moiety.
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The substrate recognition properties of the DENV protease
are well-defined. Analysis of the cleavage sites in the viral
polyprotein recognized by the NS2B-NS3 protease
demonstrates a requirement for basic residues (Arg, Lys)
particularly in the P1 and P2 positions. Small amino acids (Gly,
Ser) are commonly present in P1’ (Fig. 1).15 A wider range of
residues is tolerated in the other positions near the cleavage site.
Leu248ß
Thr179
O
1
2
3
O
A
B
OH
4
H3C
H3C O
O
O
SH
H3C
H3C
Cys241ß
Val181
Fig. 1. Cleavage site sequence logo for the flaviviral NS2B-NS3
protease, based on the six polyprotein cleavage sites from 50
different flaviviruses.15,16
Fig. 2. Structure of CA4 in the tubulin binding pocket and some of
its interactions with surrounding amino acids according to the
crystal structure by Gaspari et al. (pdb code: 5LYJ).19 The
structural elements essential for binding to tubulin are highlighted
in gray.
Since tubulin is an extensively studied target in antitumor
therapy, numerous inhibitors of tubulin polymerization have
already been synthesized and characterized, and the
biochemical properties of tubulin are well-characterized in this
context. Thus far, six distinct binding sites on tubulin for
different classes of ligands have been identified.17 A large
number of ligands is derived from the alkaloid colchicine,
which binds into a pocket that has consequently been termed
"colchicine binding site".18 Another ligand that binds into the
colchicine binding site is combretastatin A-4 (CA4; see Fig. 2),
a natural product that was originally isolated from the tree
Combretum caffrum.19 Combretastatin analogs are synthetically
accessible via the Wittig reaction of two substituted aromatic
CA4 was synthesized according to Scheme 1. The cis-
configured double bond that connects the two aromatic rings
was obtained in a Wittig reaction between the phosphonium salt
1 and isovanillin. The reaction required warming to 60°C.
Under these conditions, the main reaction product was the cis-
isomer, possibly due to the lower stability of the intermediate
betaine. Despite the slightly acidic phenolic hydroxyl group, no
protective groups were required if at least two equivalents of
base were used.
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ACS Medicinal Chemistry Letters
Ph3P
Br
+
O
1
2
3
if R = NO2:
SnCl2, NaBH4
BuLi, THF
O
R
O
A
B
R
O
A
B
NH2
-20°C --> 60°C, 12h
MeOH, RT, 24h
4
5
O
O
O
O
O
O
O
O
O
6
7
8
1
R = OH or NO2
CA4
3
R = OH:
2
R = NO2:
9
Scheme 1. Synthesis of CA4 and “combretamin” (3).
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To attach a peptide sequence that is recognized by the DENV
protease, the hydroxyl group at position B-3 was replaced by an
amino group. To obtain the derivative 3, hereafter called
“combretamin”, a nitro derivative of CA4 was synthesized (2)
and then reduced to furnish the amine. Initial attempts with
Zn/HOAc28 or H2/Pd resulted either in incomplete reduction or
hydrogenation of the double bond.
Eventually, an optimized protocol using five equivalents of
each SnCl2 and NaBH4, under absence of light and oxygen,
provided the amine 3 in quantitative yield. The oligopeptides
were synthesized by solid phase peptide synthesis (Scheme 2).
The N-terminus of the peptides was capped with a benzoyl
group to protect the otherwise free amino group and to facilitate
UV detection of the products. The coupling of the peptides to
combretamin was performed in solution, resulting in the
compounds 6a to 6h.
3
O
R2
O
R1'
O
R2
O
R1'
HATU, DIPEA, DMF
H
H
H
N
N
OH
N
N
N
N
N
0°C --> RT, 12h
B
H
H
H
H
O
R1
O
O
R1
O
O
A
O
O
R2, R1, R1' = Amino acid side chains:
R2, R1, R1' = Amino acid side chains:
O
5a
5b
6a
6b
Arg, Arg, Gly:
Arg, Arg, Ala:
Arg, Arg, Gly:
Arg, Arg, Ala:
5c
6c
Arg, Arg, ß-Ala:
Arg, Arg, ß-Ala:
5d
6d
Arg, Arg, Gly, R2'=Gly:
Arg, Arg, Gly, R2'=Gly:
5e
5f
5g
5h
6e
6f
6g
6h
Arg, Arg, Ser:
Lys, Arg, Gly:
Lys, Arg, Ser:
Lys, Arg, Ala:
Arg, Arg, Ser:
Lys, Arg, Gly:
Lys, Arg, Ser:
Lys, Arg, Ala:
Scheme 2. Synthesis of B-3-combretastatin peptide-hybrids. The expected NS2B-NS3 cleavage site is marked with a vertical
arrow.
In addition, we synthesized an analog without any prime site
residues between the cleavage site and the combretastatin
moiety. This Arg-Arg-combretamin was not hydrolyzed by the
DENV protease, but acted as an inhibitor in the biochemical
protease inhibition assay29 with a remarkably low IC50 of
6.3 µM (data not shown). Therefore, all peptide-hybrids
reported here feature one or two prime position amino acids
between the combretamin core and the Arg/Lys non-prime
residues to allow complete hydrolysis.
hydrolyzed to a significant extent by the DENV protease in
vitro. The cleavage efficiency was strongly influenced by the
P1’ residue. Alanine and glycine were tolerated well in position
P1’, but the non-proteinogenic -alanine (6c) and the polar
serine (6e, 6g) were not hydrolyzed efficiently. With respect to
the serine residue, this observation was partially unexpected
and in contrast to the prime-site preferences derived from the
viral polyprotein cleavage sites (Fig. 1).
Kinetic parameters (Km, vmax and kcat) for the hydrolysis of
the compounds were determined using the Lineweaver-Burk
plot and compared to values obtained for other published
substrates. Compound 6d had the highest affinity with a Km of
144 µM, and the Km values for 6a and 6f were around 200 µM.
The natural cleavage sites of the protease are located between
the various nonstructural proteins in the DENV polyprotein.
The first objective was to verify that an activation of the
assumed prodrugs is possible by the DENV protease in vitro.
Therefore, compounds were incubated in a solution of the
protease and the degree of conversion was quantified by HPLC-
UV. The metabolites were identified with HRMS-ESI. As
shown in Table 1, compounds 6a, 6b, 6d and 6f were
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Dodecapeptides that resemble the polyprotein cleavage sites
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Despite similar kinetic parameters, the FRET substrate was
almost completely cleaved after two hours, whereas only a
fraction of the peptide-hybrids of about 40% was hydrolyzed at
this time point (Table 1). This effect can at least partially be
explained by product inhibition. The hydrolysis product Bz-
Arg-Arg-OH (from 6a, 6b and 6d) shows 34.5% inhibition of
the DENV protease activity at 250 µM, which is the assumed
concentration of the product after half of the prodrug has been
hydrolyzed. Product Bz-Lys-Arg-OH (from 6f) inhibits 12.2%
of the protease activity at this concentration.
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were synthesized and assayed by Khumthong et al. and found
to have affinities (Km) to DENV protease in the three-digit
micromolar range as well (see supporting information).30 The
data of our compounds were also compared to the FRET
substrate that is commonly used for in vitro activity studies.29
Km values were comparable to the Km of other substrates
(Table 1). The turnover numbers (kcat) were up to 0.284 s-1 (for
compound 6b) and thus comparable to the artificial FRET
substrate with a kcat of 0.168 s-1. The resulting values were
between one and two orders of magnitude higher than for the
dodecapeptides. It is possible that the smaller size of the
peptide-hybrids and its metabolites leads to faster diffusion into
and out of the active site of the enzyme and thus to a higher
reaction rate.
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Table 1. Biochemical and antiviral data of combretastatin and its peptide-hybrids.
Tubulin
Cytotoxicity (CC50)[e]
72 h
Virus Titer
Reduction at
25 µM (24 h
in Huh-7)[d]
Hydr
Polyme
rization
Inhibiti
on %[c]
Kinetic
parameters[b]
Compd. Structure/Sequence
o-lysis
in
HeLa and
Huh-7
[a]
24 h
in
%
Huh-7
DENV:11
HeLa:
EC50=870 nM
0.428 nM
60.3
3.8
±
O
O
OH
CA4
3
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
38.3 µM
ZIKV: 70%
(@ 5 µM)
Huh-7:
1.05 nM
O
O
O
O
O
HeLa:
1.47 nM
DENV: 66%
33.1
0.9
±
±
±
NH2
> 100 µM
173 µM
123 µM
“Combr
etamin”
ZIKV: 84%
(@ 5 µM)
Huh-7:
3.93 nM
O
HeLa:
37.7 nM
O
61.0
4.9
O
O
NH NH2
4a
4b
DENV: 69%
DENV: 64%
Huh-7:
26.9 nM
O
O
O
O
O
HeLa:
6.69 nM
O
40.5
9.5
NH NH2
n.a.
Huh-7:
O
6.03 nM
Km = 224.9 µM
HeLa:
7.20 nM
Bz-Arg-Arg-Gly-
combretamin
39
2.3
±
vmax
µM/s
=
0.103 26.0
4.7
±
6a
DENV: 84%
88.6 µM
Huh-7:
kcat = 0.103 s-1
26.6 nM
Km = 949.5 µM
HeLa:
65.7 nM
Bz-Arg-Arg-Ala-
combretamin
36
0.6
±
±
vmax
µM/s
kcat = 0.284 s-1
=
0.284
6b
6c
3.4 ± 0.6 DENV: 57%
3.4 ± 2.7 DENV: 57%
111 µM
190 µM
Huh-7:
191 nM
HeLa:
952 nM
Bz-Arg-Arg-ß-Ala-
combretamin
6.7
0.8
n.d.
Huh-7:
156 nM
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Tubulin
Cytotoxicity (CC50)[e]
72 h
1
2
3
4
Virus Titer
Hydr
Polyme
rization
Inhibiti
on %[c]
Kinetic
parameters[b]
Reduction at
25 µM (24 h
in Huh-7)[d]
Compd. Structure/Sequence
o-lysis
in
HeLa and
Huh-7
[a]
24 h
in
%
Huh-7
5
6
7
8
9
Km = 144.37 µM
HeLa:
19.5 nM
Bz-Arg-Arg-Gly-Gly-
combretamin
42
4.2
±
±
±
vmax
=
0.0782 13.4
±
6d
DENV: 55%
DENV: 51%
59.7 µM
µM/s
kcat = 0.0782 s-1
7.0
Huh-7:
24.0 nM
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HeLa:
29.2 nM
Bz-Arg-Arg-Ser-
combretamin
3.4
1.7
6e
n.d.
1.9 ± 1.6
> 100 µM
> 100 µM
ZIKV: 92%
(@ 50 µM)
Huh-7:
23.1 nM
Km =172.9 µM
HeLa:
78.2 nM
Bz-Lys-Arg-Gly-
combretamin
32
3.4
vmax = 0.0357 -5.7
µM/s
kcat = 0.0357 s-1
6f
53%
±0.6
Huh-7:
172 nM
HeLa:
61.8 nM
Bz-Lys-Arg-Ser-
combretamin
6.3
1.4
±
±
-4.2
±0.5
6g
n.d.
61%
69%
77.9 µM
Huh-7:
134 nM
HeLa:
24.6 nM
Bz-Lys-Arg-Ala-
combretamin
5.2
1.4
-4.9
±4.8
6h
n.d.
> 100 µM
Huh-7:
71.6 nM
Km = 182.4 µM
FRET
2-Abz-nKRRS-(3-
94
2.0
±
vmax
=
0.168
Substrat
NO2)-Y
e
n.a.
n.a.
n.a.
n.a.
µM/s
kcat = 0.168 s-1
[a][DENV protease] = 1 µM, [compound] = 500 µM in buffer pH 9, incubated for 2 h at 37°C. Each value was determined from triplicate
wells.
[b][DENV protease] = 1 µM, [compound] from 100 µM up to1 mM in buffer pH 9, incubated for 10 min at rt. Each value was determined
from triplicate wells.
[c]Conditions: 4 mg/ml Tubulin, 1 mM GTP, 10 µM compound, 80 mM PIPES, pH 6.9, 0.5 mM EGTA, 2 mM MgCl2, 1 h, 37°C. The
given number is the mean value of two or three independent measurements and the standard deviation. Calculated is the relative
polymerization inhibition compared to an untreated (blank) measurement that counted as 0%. Negative numbers indicate increased
polymerization.
[d]Virus titer reduction was measured in a single point experiment. The virus containing supernatant was aliquoted into duplicates; after
incubation the average number of plaques was counted and the reduction was calculated in comparison to an untreated blank (see Table S3
in the SI).
[e]The cytotoxic concentration (CC50) was determined after 24 h incubation in Huh-7 cells and after 72 h in HeLa and Huh-7 cells. Each
value was determined from triplicate wells; the standard deviation was always below 10%.
n.a. = not applicable (no hydrolysis site or no tubulin ligand)
The second objective in creating compounds that could act as
prodrugs was to decrease the cytotoxicity, relative to CA4,
while maintaining antiviral activity. The cytotoxic
concentration (CC50) was determined after 24 h and after 72 h
of incubation in HeLa and Huh-7 cells. Like other inhibitors of
tubulin polymerization, CA4 is a highly toxic molecule that
inhibits mitosis, which eventually leads to apoptosis. After
24 h, however, mitosis has been initiated only in a fraction of
cells, so the acute toxicity is moderate. The empirical doubling
time is ca. 24 h for HeLa cells and ca. 48 h for Huh-7 cells.
Consequently, the full toxic effect becomes evident only after
extended incubation times. The readout for the antiviral effect
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(reduction of DENV and ZIKV titer in cells) was initiated after
24 h, and the antiviral activity was studied at compound
concentrations lower than the CC50 (determined after 24h).
recognition properties between the isolated NS2B-NS3 protein
construct and the intracellular protease in the DENV2proHeLa
cells, or by a combination thereof.
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2
3
The ability of CA4 and its derivatives to inhibit the
polymerization of tubulin was determined with the isolated
protein in vitro. The highest inhibitory effect was observed for
CA4, which reduced the polymerization of tubulin by 60.3% at
a concentration of 10 µM. In contrast, the peptide-hybrids had
almost no effect on tubulin polymerization (< 10% inhibition).
This indicates that, as intended, the bulky peptides attached to
the combretastatin effectively prevent these compounds from
binding into the tubulin subunit.
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The reference compound CA4 had also the strongest
cytotoxic effect, both after 24 h and 72 h (Table 1). Its effective
antiviral concentration against DENV was 870 nM in Huh-7
cells as reported earlier.11 Combretamin 3 showed similar
antiviral effect against ZIKV, yet slightly lower anti-DENV
activity and cytotoxicity. Despite the residual amino acids at
position B-3, the primary metabolites 4a and 4b showed tubulin
polymerization inhibition, cytotoxicity and antiviral activity. It
can be hypothesized that the residual amino acid is hydrolyzed
intracellularly by host enzymes, such as exopeptidases from the
cathepsin family.15 For example, cathepsin H, with preference
for Arg-containing sequences, has been isolated from
hepatocytes31 and is abundant in endosomes.32
Fig. 3. Relative fold change in CC50 between wildtype HeLa and
the DENV2proHeLa cells, which contain an intracellular DENV
protease. Numerical cytotoxicity values are provided in Table S4
in the supporting information.
The lack of correlation between the biochemical effects
(cleavage by isolated DENV protease and effect on tubulin
polymerization) and the inhibition of viral replication can be
due to three factors: i) Cleavage of the prodrugs by non-viral
intracellular proteases such as cathepsins or prohormone
convertases. The extensive cleavage of compounds such as 6d
and 6e by trypsin (cf. Table S2 in the SI) indicates that the
prodrugs possess a certain degree of promiscuity towards non-
flaviviral proteases. ii) Pharmacokinetic effects, particularly a
preferential or hindered uptake of prodrugs depending on the
peptidic substituent. The mostly basic amino acids in the
prodrug moiety are likely to favor, to a variable degree, binding
to negatively charged cell membranes and subsequent cellular
internalization by endocytosis. iii) Involvement of as-yet
undefined targets or mechanisms in the expression of the
phenotypic effect of these compounds.
The design goals were achieved in that all peptide-hybrids
had a clearly reduced toxicity compared to CA4, being about
20 to 2000 times less toxic in the 72 h measurement. All peptide
hybrids (6a–h) were further tested in a virus titer reduction
assay and showed an antiviral effect in the low micromolar
range. It is noticeable that the compounds which did not
undergo hydrolysis by DENV protease in vitro still have
antiviral activity. Both findings can be explained by the fact that
these compounds may also be inhibitors of the viral protease.
The IC50 of compound 6e was exemplary determined to be
25 µM (see SI). As already outlined above, other intracellular
proteases may cleave this compound, delivering a CA4-
derivative in the cell.
In order to confirm proteolytic cleavage and prodrug
activation by the DENV protease, we employed HeLa cells
stably transfected with DENV serotype 2 NS2B and NS3
proteins (DENV2proHeLa). Compounds that are hydrolyzed by
the viral protease are expected to show increased cytotoxicity
in these cells as compared to wildtype HeLa cells. In contrast,
non-hydrolyzable compounds are expected to have similar
effects in both cell lines. As shown in Figure 3, the reference
compounds CA4 and 3 and the non-cleavable compounds 6c,
6e and 6g have practically identical toxicity in both cell lines.
In contrast, two of the four hydrolyzable compounds (6a, 6b)
In conclusion, combretastatin peptide-hybrids have been
synthesized and found to be hydrolyzed by the DENV protease
in vitro at a rate comparable to its natural substrates. Peptide-
hybrids and metabolites showed antiviral activity against
DENV and ZIKV in hepatocyte-derived cells at subcytotoxic
concentrations. However, not all assumed prodrugs were
hydrolyzed, the SAR does not provide a clear relationship
between prodrug activation and antiviral efficacy, and the
therapeutic index is insufficient to consider further preclinical
development. We therefore consider the present work as a
conceptual study that sheds more light on the antiviral activity
of tubulin inhibitors, especially from the combretastatin group,
and prodrugs activated by viral factors.
show
a
remarkable increase of cytotoxicity in the
DENV2proHeLa cells, which we regard as an indication of
intracellular cleavage and proof-of-concept for the prodrug
approach presented here. The relatively weak and probably
insignificant increase of cytotoxicity for 6d and 6f can be
caused by insufficient permeability, by variations of substrate
ASSOCIATED CONTENT
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ACS Medicinal Chemistry Letters
Broad-Spectrum Agents for Flaviviral Infections: Dengue, Zika
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AUTHOR INFORMATION
Corresponding Author
*E-mail: c.klein@uni-heidelberg.de
ORCID
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Michael Richter: 0000-0002-8227-0543
Mila M. Leuthold: 0000-0001-7698-2360
Dominik Graf: 0000-0002-6124-8451
Ralf Bartenschlager: 0000-0001-5601-9307
Christian D. Klein: 0000-0003-3522-9182
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Author Contributions
All authors have given approval to the final version of the
manuscript.
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ACKNOWLEDGMENT
We thankfully acknowledge the technical assistance of Stephanie
Kallis for the ZIKV antiviral assay, Natascha Stefan for the help in
cell culture, Tobias Timmermann and Heiko Rudy for NMR and
mass spectral data, Miriam Jaki and Irene Martinez for assistance
in synthesis and Veaceslav Boldescu for support with the tubulin
assay. All persons are associated with Heidelberg University,
IPMB Medicinal Chemistry.
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ABBREVIATIONS
CA4, Combretastatin A-4; DENV, dengue virus; DIPEA,
diisopropylethylamine; DMF, dimethylformamide; EGTA,
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid;
HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxide hexafluorophosphate; NS 3, nonstructural
protein 3; PIPES, 1,4-Piperazinediethanesulfonic acid; SAR,
Structure-Activity-Relationship; ZIKV, Zika virus.
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Steinmetz, M. O.; Prota, A. E. Microtubule-Targeting Agents:
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Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar,
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