Exploring the Implication of DDX3X in DENV Infection: Discovery of the First-in-Class DDX3X Fluorescent Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.9b00681

In the absence of effective drugs or vaccines for the treatment of the five Dengue Virus serotypes, the search for novel antiviral drugs is of primary importance for the scientific community. In this context, drug repurposing represents the most used strategy; however, the study of host targets is now attracting attention since it allows identification of broad-spectrum drugs endowed with high genetic barrier. In the last ten years our research group identified several small molecules DDX3X inhibitors and proved their efficacy against different viruses including novel emerging ones. Herein, starting from a screening of our compounds, we designed and synthesized novel derivatives with potent activity and high selectivity. Finally, we synthesized a fluorescent inhibitor that allowed us to study DDX3X cellular localization during DENV infection in vitro. Immunofluorescence analysis showed that our inhibitor colocalized with DDX3X, promoting the reduction of infected cells and recovering the number of viable cells.

Full text of DOI:10.1021/acsmedchemlett.9b00681

pubs.acs.org/acsmedchemlett
Letter
Exploring the Implication of DDX3X in DENV Infection: Discovery of
the First-in-Class DDX3X Fluorescent Inhibitor
Annalaura Brai,∇ Adele Boccuto,∇ Martina Monti, Serena Marchi, Ilaria Vicenti, Francesco Saladini,
Claudia Immacolata Trivisani, Alessandro Pollutri, Claudia Maria Trombetta, Emanuele Montomoli,
Valentina Riva, Anna Garbelli, Emanuele Maria Nola, Maurizio Zazzi, Giovanni Maga, Elena Dreassi,*
and Maurizio Botta
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ABSTRACT: In the absence of effective drugs or vaccines for the
treatment of the five Dengue Virus serotypes, the search for novel
antiviral drugs is of primary importance for the scientific
community. In this context, drug repurposing represents the
most used strategy; however, the study of host targets is now
attracting attention since it allows identification of broad-spectrum
drugs endowed with high genetic barrier. In the last ten years our
research group identified several small molecules DDX3X
inhibitors and proved their efficacy against different viruses
including novel emerging ones. Herein, starting from a screening
of our compounds, we designed and synthesized novel derivatives
with potent activity and high selectivity. Finally, we synthesized a
fluorescent inhibitor that allowed us to study DDX3X cellular
localization during DENV infection in vitro. Immunofluorescence analysis showed that our inhibitor colocalized with DDX3X,
promoting the reduction of infected cells and recovering the number of viable cells.
KEYWORDS: DDX3X, host factors, DENV, antiviral agents, resistance, immunofluorescence
engue Virus (DENV) is a positive sense, single stranded
virus member of the Flaviviridae family. According to
the search for novel effective antiviral drugs is an important
challenge for the scientific community. Our research group
already proved that the ATPase/RNA helicase X-linked
DEAD-box polypeptide 3 (DDX3X) is an important target
to develop antivirals with indirect acting mechanism of
action.4−9 In 2016 we reported the first DDX3X inhibitor
with a urea-based scaffold, endowed with broad spectrum
antiviral activity against DENV, West Nile virus (WNV),
Hepatitis C virus (HCV), and immunodeficiency virus type 1
(HIV-1).7 Furthermore, we discovered a sulfonamide series of
DDX3X inhibitors with promising activities and improved in
vitro pharmacokinetic properties, which were active against
WNV infection.8 DDX3X inhibitors offer multiple advantages,
in particular the possibility to fight different viruses including
novel emerging ones with a unique molecule, and the reduced
risk for drug resistance, since human genes coding for proteins
D
World Health Organization data, the global incidence of the
five serotypes of DENV (DENV1−5) has increased in recent
years, and about half of the world’s population is now at risk of
infection.1 Nowadays DENV is transmitted in over 128
countries by Aedes aegypti and Aedes albopictus infected
mosquitoes, with 390 million new cases per year.1 The
human migration flows, the inadequate vector control, and the
global warming are the possible causes of the rapid spread of
this virus. DENV infection is often asymptomatic or causes flu-
like symptoms, such as high fever, that usually terminate after 1
week. However, in a small number of cases DENV infection
results in a potentially deadly illness named Dengue
hemorrhagic fever (DHF) or in Dengue shock syndrome
(DSS), characterized by high fever, bleeding, and circulatory
failure which can cause shock and sometimes death.2 No
specific treatments are actually available, and vaccination is
serotype-specific. In 2015 Sanofi Pasteur produced the first
dengue vaccine, Dengvaxia, now approved for use in endemic
areas.3 Nevertheless, its use is strictly recommended only to
the patients that already contracted DENV infection, since the
vaccination can increase the risk of contracting more severe
DENV with respect to unvaccinated patients.3 In this context,
Special Issue: In Memory of Maurizio Botta: His
Vision of Medicinal Chemistry
Received: December 31, 2019
Accepted: March 2, 2020
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© XXXX American Chemical Society
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Letter
involved in viral replication are minimally vulnerable to
mutations induced by drug administration. The implication
of DDX3X in the life cycle of DENV is still debated in the
literature, and different studies describe DDX3X either as a
pro-viral or as an antiviral protein.10−12 Indeed, Kumar et al.
reported that DDX3X knock down leads to increased viral
titers;10 in contrast, Khadka and co-workers showed that
knockdown of DDX3X reduces the expression of DENV
reporter gene, thus highlighting a negative role in DENV
replication.11
quantity of cellular ATP. According to the reported mechanism
of toxicity, the half maximal cytotoxic concentrations (CC50)
of compounds decreased with respect to those previously
obtained using the MTT assay in Huh-7 cells. Nonetheless, all
compounds showed selectivity indexes (SI) higher than ten,
with the exception of 2 (SI = 3). Among known DDX3X
inhibitors (Chart 1), compound 5 exhibited the most
promising anti-WNV activity and resulted to be the most
active of the series also against DENV infection with an IC50 of
1.6 μM and low cytotoxicity (SI = 94).8 The trifluoromethyl
derivative 2 presented lower antiviral activity and higher
toxicity. Compounds 3 and 4 had antiviral activities in the low
micromolar range and low toxicity similarly to compound 5,
while methoxy derivative 6 shows low antiviral activity and
higher standard deviation, probably due to its lower solubility.
In order to investigate the cellular mechanisms of our
inhibitors during viral replication, we analyzed the effects of
compound 3 in the number of viable cells, in the expression of
viral dsRNA and DENV not structural proteins 3 and 5 (NS3
and NS5). Huh-7 cells were infected with DENV-2 strain at
the concentration of 100 TCID50. Cells were treated with
increasing concentrations of compound 3 (0.1, 1, 10, 20 μM),
and at 72 h post infection (p.i.) cells were fixed, stained with
specific fluorescent antibodies, and analyzed by confocal
microscopy.
As reported in Figure 1, compound 3 reduced the number of
NS3 (white bars), NS5 (blue bars), and viral dsRNA (yellow
bars) positive cells. In addition, the treatment with compound
3 was associated with a higher number of viable cells,
contrasting the cytopathic effect associated with DENV-2
infection. Taking into account these results, extensive
molecular dynamic studies were carried out to design a small
series of derivatives (for details see Supporting Information).
The novel urea and sulfonamide compounds 7−24 were thus
designed and synthesized. Full synthetic protocols are
described in the Supporting Information. Compounds were
tested for their ability to inhibit the helicase activity of DDX3X
using our FRET-based biochemical assay previously pub-
lished.5,7,8 As reported in Chart 2, several derivatives showed
activities from the low micromolar to the submicromolar
range. As suggested by docking and molecular dynamic studies,
the replacement of methyl with small and electron withdrawing
groups such as fluorine is well tolerated in ortho (7 and 11)
and in meta positions (compounds 8, 9, and 10), while the
para substituted derivative 12 is about ten-times less active. In
contrast with the sulfonamide series the ethoxymethyl
introduction was less tolerated than the other substitutions,
with compound 9 being about 100-times less active. Taking
into account our previously published studies7,8 the butyl side
chain on the triazole ring was replaced with other linear and
not hindered substituents, such as isopentyl and 3-oxobutyl.
The replacement of butyl with oxobutyl slightly decreased the
activity (compound 10); in contrast, the substitution with
isopentyl led to compounds with comparable activity (11 and
8). The methyl moiety was replaced with the bioisosteric
trifluoromethyl group, and as already observed for the
sulfonamide series it was well tolerated (compounds 13, 14,
16−18, and 20). Among the series of trifluoromethyl
derivatives, 13 with a methoxy group in the meta position
with respect to the triazole ring was less active than the
corresponding ortho derivative 14, with 14 being able to form
an additional hydrogen bond between the oxygen of the
methoxy group and Gly325. Since bulky electron donating
Since an X-ray crystal structure of DDX3X in its active
closed conformation was disclosed only very recently,13 but
was unavailable at the start of this study, we previously
generated a homology model that led us to identify two series
of RNA-competitive inhibitors.5,7 Compound 1 with a urea-
based structure was characterized by a promising broad-
spectrum antiviral activity, good passive permeability, and
metabolic stability but limited aqueous solubility. On the
contrary the sulfonamide series was characterized by higher
solubility values and improved activity against WNV with
respect to the hit compound 1 (compounds 2−6).8 In order to
verify their activity against DENV, the most promising
sulfonamide compounds were assayed against DENV-2
through an immunodetection assay. Briefly, human hepatoma
cells (Huh-7) were infected with 50 TCID50 of DENV-2 strain
in the presence of serial dilution of compounds. After 72 h,
supernatants from each well were used to infect preseeded
Huh-7 cells in a second cycle of infection. Viral replication was
determined after 72 h by an immunoenzymatic assay using a
monoclonal antibody targeting a conserved region of viral E
antigen. The measurement of the half maximal inhibitory
concentration (IC50s) indicated that most of the compounds
had an antiviral activity in the low micromolar range,
comparable or lower than the activity of the already described
hit compound 1 (Chart 1). Since DDX3X inhibitors were
demonstrated to interfere with mitochondrial translation
causing lower intracellular ATP concentrations,14 we replaced
the MTT assay with a CellTiter-Glo 2.0 (Promega) kit, which
determines the number of viable cells by measuring the
Chart 1. Structures of Known DDX3X Inhibitors, Anti-
Enzymatic Activities Expressed as Half Maximal Inhibitory
Concentrations (IC50) Standard Deviation (SD),
Antiviral Activities against DENV-2, and Cytotoxicity on
Huh-7 Cells*
*
Previously published data.
B
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Figure 1. Effects of compound 3 on the viral replication of DENV-2. As reported in the panels, compound 3 reduced the number of NS3 (white
bars), NS5 (blue bars), and viral dsRNA (yellow bars) positive cells. Compound 3 had a protective effect and recovered the number of viable cells
(red bars, bottom panel).
groups were well accepted in the sulfonamide series,8 we
synthesized an ortho methylsulfonamido derivative in which
ethoxymethyl was replaced with a butyl side chain. As a result
15 showed a high inhibitory activity with an IC50 value of
about 0.1 μM. Compound 16 with a fluoro-methyl substitution
at C(4) triazole position was about ten-times less active than
derivatives 19 and 20, confirming that longer lipophilic side
chains are preferred; in contrast, derivative 17 was about 20-
times less active, probably due to the perfluorobutyl side chain,
which according to docking analysis is accommodated outside
the binding site.
The sulfonamide series demonstrates promising antienzy-
matic activities, with compounds 21, 22, and 24 being
characterized by activities of 0.06, 0.005, and 0.08 μM.
Morpholino derivative 21 maintains all the key interactions of
the sulfonamide series, in particular a hydrogen bond between
triazole and Arg276 and two hydrogen bonds between
sulfonamide and Arg480 and Arg276, and establishes an
additional hydrogen bond between the morpholine oxygen and
Arg351. Ester 22 was extremely active; however, the
corresponding carboxylic acid 23 was not evaluated due to
its precipitation during the assays. Finally, an ortho
methylsulfonamido derivative was synthesized (24); its activity
value of 0.08 is comparable to that of urea compound 15. Even
in this case, 24 maintains all the key interactions and forms an
additional hydrogen bond between the oxygen of sulfonamide
and Arg351.
The antiviral activity and cytotoxicity of 12 selected
compounds were tested using the immunodetection assay as
described in the Supporting Information. Their IC50s values
were in the low micromolar to submicromolar range and were
comparable to or lower than those of ribavirin and sofosbuvir,
two broad spectrum antivirals used in our tests as reference
compounds.15,16 As shown in Table 1, the best result was
represented by compound 7, characterized by an IC50 of 0.9
μM and low cytotoxicity, with its selectivity index being equal
to 222. Compound 9 was found inactive probably due to its
lower DDX3X inhibition, while derivative 10 had a very
promising IC50 value of 0.3 μM and a selectivity index of 19.
Fluorinated derivative 11 showed high selectivity index, but it
was about four-times less active than the corresponding
isopentyl derivative 8.
Compounds 19 and 20 had favorable antiviral activities and
cytotoxicity, comparable to those of the fluorinated compound
8. Sulfonamide 21 and 22, despite their potent DDX3X
inhibitory concentrations showed activities of 5.3 and 4.3 μM,
respectively. The low activity of 22 is probably due to its
cellular hydrolysis into the corresponding carboxylic acid 23,
which was found toxic and inactive. Sulfonamide 24 and urea
15 showed IC50 of 2.5 and 8.3 μM, respectively, and their
selectivity indexes were lower than those of the other
compounds of the series.
The exact implication of DDX3X in the life cycle of
flaviviruses such as WNV or DENV is actually poorly
understood. Nevertheless, in the last few years, immuno-
fluorescence studies have been performed by different groups
to investigate the DDX3X role during WNV17 and JEV18
infection by analyzing its colocalization with cellular and viral
proteins.
In order to better understand the mode of action of our
compounds in DENV infected cells, we planned the synthesis
of a fluorescent inhibitor to be used as a probe in time course
confocal microscopy experiments. Fluoresceine isothiocyanate
(FITC) was selected as fluorophore; computational analysis
led us to choose sulfonamide 3 for further modifications and
the side chain on C (4) triazole position as an optimal position
to insert FITC. The synthesis of the fluorescent probe (25)
was accomplished according to Scheme 1, and first entailed the
synthesis of azide 29. Sulfonamide 28 was synthesized starting
from 2-trifluoromethylaniline 26 and 3-nitrosulfonide chloride
27. Subsequent Pd on charcoal catalyzed hydrogenation
furnished the corresponding aniline that was converted into
azide 29 through diazotization reaction.8 Alkyne 32 was
synthesized starting from ethanolamine 30, that was protected
with di-tert-butyl dicarbonate and converted into the terminal
alkyne 31 by reaction with propargyl bromide. Acidic
deprotection of the amino group and reaction with FITC
furnished the fluorescent alkyne 32. Click reaction between
C
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Chart 2. Structures of Novel DDX3X Inhibitors
Synthesized, Anti-enzymatic Activities Expressed as Half
Maximal Inhibitory Concentrations (IC50), Calculated
Using a FRET-Based Assay
Table 1. Antiviral Activity of Selected Compounds against
DENV-2 Infected Cells
a
c
b
c
Cpd ID
IC50 SD (μM)
CC50
SD (μM)
200 12
SI
7
8
9
0.9 0.1
2.5 0.8
>145
222.2
80.0
200 23
145 18
10
11
15
19
20
21
22
23
0.3 0.1
10.5 9.1
2.5 0.2
1.4 0.8
2.7 0.1
5.3 5.0
4.3 2.3
>7
20
200 18
35
170 24
170 19
4
64.7
19.0
14.0
121.4
62.9
18.9
23.2
6
100
100 14
7.0
9
2
24
8.3 3.7
4.0 0.6
3.8 1.1
87 11
100 14
200 17
10.5
25.0
52.6
ribavirin
sofosbuvir
a
IC50: mean
standard deviation of half maximal inhibitory
concentration calculated in Huh7 cells from at least two experiments;
b
CC50: half maximal cytotoxic concentration, evaluated on Huh-7
c
cells. SI: Selectivity index, calculated as the ratio between CC50 and
IC50.
helicase family as DDX3X. Thus, proper assembly of these
subcellular structures has functional consequences for DENV
replication and infectivity.20,21 Moreover, our analysis showed
that DDX3X increases its expression during infection, with a
maximum expression at 48 and 72 h (CV panels in Figure S1).
As shown in Figure 2 (left panels), 25 colocalized with
DDX3X at the perinuclear region in the first 6 h of treatment
and then induced DDX3X cytoplasmatic localization as in
uninfected Huh7 cells (Figure S1, CC panel). The same
experiment was performed by analyzing cellular localization of
the viral protein NS5 using a specific antibody detected using
Alexaflour-labeled secondary antibody (Figure 2, right panels).
In the first 6 h, NS5 had a cytoplasmatic localization and was
colocalized with 25 and then moved in the nucleus. As shown
in Figure S2 and Figure S3, 25 was associated with an
increased number of viable cells and with a significant
reduction of the total number of NS5 positive cells at 48
and 72 h.
In the present work, we focused our efforts in expanding the
structure activity relationship (SAR) around the two series of
already discovered DDX3X inhibitors, concentrating our work
on the search of novel promising compounds active against
DENV infection. As a result, we discovered novel DDX3X
helicase inhibitors with improved antiviral activity, comparable
to or lower than those reported for known broad spectrum
antivirals such as ribavirin or sofosbuvir. Notably, the most
promising derivative, compound 10, is about 9-times more
active than the previous hit (compound 1). In addition, we
investigated the mechanism of action of our compounds in
infected cells, using the novel fluorescent derivative 25.
Immunofluorescence analysis confirms that 25, during the
first hours of DENV infection, colocalized with DDX3X,
promoting the reduction of NS5 positive cells and recovering
the cell number, over time (until 72 h).
azide 29 and terminal alkyne 32 led to the desired FITC-
labeled derivative 25.
Compound 25 was tested for its ability to inhibit DENV-2
replication in Huh-7 cells, its IC50 was 28.2 4.6 μM, and its
CC50 was 140.0 μM. Starting from this result, we studied the
time course localization of our fluorescent probe investigating
its colocalization with DDX3X and viral protein NS5. Huh-7
cells were infected with DENV-2 at the TCID50 of 100, fixed at
0, 6, 12, and 24 h.p.i., permeabilized and immunostained.
Confocal microscopy analysis revealed that DDX3X is
primarily localized into the cytoplasm, while during viral
infections DDX3X is recruited to perinuclear spots, particularly
between 6 and 24 h.p.i. (Figure 2 and Figure S1). The
recruitment of DDX3X to these structures could be a potential
shared strategy employed by RNA viruses, such as HCV and
WNV, that exploit DDX3X function.17−19 Similarly, DENV
interacts with other components within stress granules and
processing bodies sites, such as DDX6, a member of the same
The low cytotoxicity of compounds, evaluated by measuring
ATP concentration, indicates once again that our compounds
are characterized by high cellular tolerability. Overall, results
reported herein confirm that DDX3X inhibitors represent a
D
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a
Scheme 1. Synthesis of the Fluorescent Inhibitor 25
a
Reagents and conditions: (i) Pyr, 5 h, r.t. (ii) (a) H2, Pd/C, MeOH, 2 h; (b) t-BuONO, CH3CN, 20 min. 0 °C; (c) TMSN3, CH3CN, 2 h, r.t.;
(iii) (a) Di-tert-butyldicarbonate, DCM, 3 h, 0 °C to r.t.; (b) NaH, DMF, 0 °C to r.t., then propargyl bromide, 9 h, r.t.; (iv) (a) HCl (3 N), MeOH,
r.t., 2 h; (b) TEA, DCM, 1 h, r.t., then FITC, 12 h, r.t.; (v) CuSO4·5H2O, sodium ascorbate, H2O, t-BuOH (1:1), MW, 300 W, 10 min, 120 °C.
Figure 2. Immunofluorescence analysis. DENV-2 infected cells were treated with DDX3X fluorescent inhibitor 25 at a concentration of 20 μM.
Cells were stained at different time-points with a DDX3 antibody (detected using Alexaflour568 labeled secondary antibody, left panels) and
DENV NS5 antibody (detected using Alexaflour (568) labeled secondary antibody, right panels) and examined by confocal microscopy. Individual
antibody stained as well as merged images are shown as indicated. Each experiment was repeated at least two times.
safe and promising class of antivirals, supporting their
evaluation in an animal model of DENV infection.
Authors
Annalaura Brai − Dipartimento Farmaco Chimico Tecnologico,
Universita degli Studi di Siena, 53100 Siena, Italy;
orcid.org/0000-0001-6395-9348
Adele Boccuto − Dipartimento di Biotecnologie Mediche,
Universita degli Studi di Siena, 53100 Siena, Italy
Martina Monti − Dipartimento di Medicina Molecolare e dello
Sviluppo, Universita degli Studi di Siena, 53100 Siena, Italy;
orcid.org/0000-0002-2173-7269
Serena Marchi − Dipartimento di Medicina Molecolare e dello
Sviluppo, Universita degli Studi di Siena, 53100 Siena, Italy
Ilaria Vicenti − Dipartimento di Biotecnologie Mediche,
Universita degli Studi di Siena, 53100 Siena, Italy
Francesco Saladini − Dipartimento di Biotecnologie Mediche,
Universita degli Studi di Siena, 53100 Siena, Italy;
orcid.org/0000-0002-9934-377X
Claudia Immacolata Trivisani − Dipartimento Farmaco
Chimico Tecnologico, Universita degli Studi di Siena, 53100
̀
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00681.
̀
̀
Full synthetic procedures and spectral characterization,
data for all intermediates and final compounds,
experimental details of the enzymatic assays, antiviral
assays, the cytotoxicity experiment, and the immuno-
fluorescence experiments (PDF)
̀
̀
̀
AUTHOR INFORMATION
■
Corresponding Author
̀
Elena Dreassi − Dipartimento Farmaco Chimico Tecnologico,
̀
Universita degli Studi di Siena, 53100 Siena, Italy;
Siena, Italy
orcid.org/0000-0001-8987-940X; Email: elena.dreassi@
unisi.it
Alessandro Pollutri − Dipartimento Farmaco Chimico
Tecnologico, Universita degli Studi di Siena, 53100 Siena, Italy
̀
E
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Claudia Maria Trombetta − Dipartimento di Medicina
isothiocyanate; TCID50, half maximal tissue culture infectious
dose; DENV, Dengue Virus
̀
Molecolare e dello Sviluppo, Universita degli Studi di Siena,
53100 Siena, Italy
REFERENCES
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■
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○Maurizio Botta − Dipartimento Farmaco Chimico
̀
Tecnologico, Universita degli Studi di Siena, 53100 Siena, Italy;
Sbarro Institute for Cancer Research and Molecular Medicine,
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00681
(7) Brai, A.; Fazi, R.; Tintori, C.; Zamperini, C.; Bugli, F.;
Author Contributions
́
Sanguinetti, M.; Stigliano, E.; Este, J.; Badia, R.; Franco, S.; et al.
Annalaura Brai designed and synthesized compounds and
wrote the original manuscript. AP synthesized compounds.
Adele Boccuto, IV, and FS performed DENV and cytotoxicity
assays, VR, AG, and EMN purified DDX3X and performed
enzymatic assays. MM, SM, and CT performed immuno-
fluorescence assays. CIT performed in silico studies. ED
analyzed compounds. MZ supervised antiviral and cytotoxicity
assays. EM supervised immunofluorescence assays. GM
supervised enzymatic assays. GM and MB had the original
idea. MB supervised the project.
Human DDX3 Protein Is a Valuable Target to Develop Broad
Spectrum Antiviral Agents. Proc. Natl. Acad. Sci. U. S. A. 2016, 113
(19), 5388−5393.
(8) Brai, A.; Martelli, F.; Riva, V.; Garbelli, A.; Fazi, R.; Zamperini,
C.; Pollutri, A.; Falsitta, L.; Ronzini, S.; Maccari, L.; et al. DDX3X
Helicase Inhibitors as a New Strategy To Fight the West Nile Virus
Infection. J. Med. Chem. 2019, 62 (5), 2333−2347.
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Author Contributions
∇Annalaura Brai and Adele Boccuto equally contributed to this
work.
Notes
The authors declare no competing financial interest.
○Deceased on August 2, 2019.
ACKNOWLEDGMENTS
■
We are grateful to First Health Pharmaceuticals B.V., the
exclusive license holder of the DDX3 inhibitors discussed in
this paper, for their economic support of MB, MZ, FS, and
ABo. Financial support was also provided by Tuscany Region
(DD3242/2009 Bando Salute 2009) to MB. GM and VR were
partially supported by the CNR project “Identification of novel
therapeutic molecules against Zika and other emerging viruses.
́ ́ ́
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DEDICATION
■
This work is dedicated to Professor Maurizio Botta, who
transmitted his devotion for science to generations of
researchers, extending, during the years, his critical but hopeful
vision of medicinal chemistry.
ABBREVIATIONS
TMSN3, trimethylsilylazide; tBuONO, tert-butylnitrite; FRET,
fluorescence resonance energy transfer; FITC, fluoresceine
■
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