Tài liệu Báo cáo khoa học: Progress for dengue virus diseases Towards the NS2B–NS3pro inhibition for a therapeutic-based approach ppt

REVIEW ARTICLE
Progress for dengue virus diseases
Towards the NS2B–NS3pro inhibition for a therapeutic-based
approach
Sonia Melino and Maurizio Paci
Department of Chemical Science and Technology, University of Rome ‘Tor Vergata’, Italy
One hundred million cases of dengue fever (DF) are
estimated by the World Health Organization to
occur yearly, together with between 250 000 and
500 000 cases of dengue hemorrhagic fever (DHF).
Extensive plasma leakage in various serous cavities
of the body, including the pleura, the pericardium
and the peritoneal cavities, may result in profound
shock, the so-called dengue shock syndrome (DSS).
The case ⁄ fatality rate of DHF in most countries is
about 5%, although appropriate symptomatic treat-
ment has been successful in reducing the mortality
of DHF to less than 1%. Most fatalities occur
among children and young adults. DF and DHF are
primarily diseases of tropical and subtropical areas,
but represent a typical example of a global disease.
The transmission of dengue virus (DENv) has
Keywords
dengue hemorrhagic fever; dengue virus;
NS3; protease inhibitors; vaccines; viral
diseases; viral serine protease
Correspondence
S. Melino, Dipartimento di Scienze e
Tecnologie Chimiche, Universita
`
di Roma

‘Tor Vergata’ via della Ricerca Scientifica,
00133 Rome, Italy
Fax: +39 0672594328
Tel: +39 0672594449
E-mail:
(Received 22 January 2007, revised 16
March 2007, accepted 17 April 2007)
doi:10.1111/j.1742-4658.2007.05831.x
Transmitted by the Aedes aegypti mosquito, the dengue virus is the etiolog-
ical agent of dengue fever, dengue hemorrhagic fever and dengue shock
syndrome, and, as such, is a significant factor in the high death rate found
in most tropical and subtropical areas of the world. Dengue diseases are
not only a health burden to developing countries, but pose an emerging
problem worldwide. The immunopathological mechanisms appear to
include a complex series of immune responses. A rapid increase in the lev-
els of cytokines and chemical mediators during dengue disease plays a key
role in inducing plasma leakage, shock and hemorrhagic manifestations.
Currently, there are no vaccines available against dengue virus, although
several tetravalent live-attenuated dengue vaccines are in clinical phases I
or II, and prevention through vaccination has become a major priority on
the agendas of the World Health Organization and of national ministries
of health and military organizations. An alternative to vaccines is found in
therapeutic-based approaches. Understanding the molecular mechanisms of
viral replication has led to the development of potential drugs, and new
molecular viral targets for therapy are emerging. The NS3 protease domain
of the NS3 protein is responsible for processing the viral polyprotein and
its inhibition is one of the principal aims of pharmacological therapy. This
review is an overview of the progress made against dengue virus; in partic-
ular, it examines the unique properties – structural and functional – of the
NS3 protease for the treatment of dengue virus infections by the inhibition

of viral polyprotein processing.
Abbreviations
ADE, antibody-dependent enhancement; DENv, Dengue virus; DF, dengue fever; DHF, dengue hemorrhagic fever; DSS, dengue shock
syndrome; E protein, glycoprotein E; ER, endoplasmic reticulum; HCV, hepatitis C virus; NS, nonstructural; NS3pro, NS3 protease domain;
NTPase, nucleotide three phosphate hydrolase; protein C, nucleocapsid protein C; prM, protein M.
2986 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS
increased considerably in recent years as a result of
the expansion of the Aedes aegypti mosquito to dif-
ferent geographic areas, and DHF has spread from
South East Asia to the Western Pacific and the
Americas. A substantial number of people travelling
to endemic regions are also infected each year. In the
last year, DHF has again been on the increase in
India and in several Asian countries because of sea-
sonal factors. Dengue is one of the most important
mosquito-borne viral diseases affecting humans; its
global distribution is comparable to that of malaria
and an estimated 2.5 billion people live in areas at
risk from epidemic transmission (Fig. 1). In 1906,
Bancroft published the first evidence implicating the
mosquito A. aegypti as the vector of DENv [1].
DENv was originally classified as an arthropod-borne
animal virus (arbovirus). The arboviruses comprise
infection agents that are biologically transmitted
between susceptible hosts by hematophagous arthro-
pods and are classified in different virus families
according to viral genes, virion structure and the viral
replication cycle. DENv belongs to the Flavivirus
genus of the family Flaviviridae that are members of
the positive-stranded virus supergroup 2 [2].

The normal cycle of DENv infection is considered
to be human–mosquito–human. Feeding on an infected
and viremic human enables the female mosquito to
transmit the virus after an incubation period of
8–10 days, during which DENv infection, replication
and dissemination results in the infection of salivary
glands. The mosquito is able to transmit DENv for its
entire life. The DENv is transmitted to a person when
an infected mosquito introduces anticoagulant sub-
stances, present in its saliva to prevent the recipient’s
blood clotting, during feeding.
Four antigenically distinct members of the DENv
serotype complex have been identified (DEN-1, DEN-
2, DEN-3 and DEN-4), and these are considered as
four distinct species belonging to the mosquito-borne
cluster clade IX of Flavivirus [2]. The corresponding
viruses of the four serotypes are genetically closely
related to one another. All DENv serotypes can cause
DF, a mild self-limiting acute febrile illness, but 1–5%
of patients with DF may experience more complicated
and severe diseases, such as DHF and DSS [3,4]. Infec-
tion with one of these four DENv serotypes provides
immunity to only that serotype for life; therefore, per-
sons living in a dengue-endemic area can have more
Fig. 1. Map of the countries of the world at risk of dengue virus infections in 2006, according to the World Health Organization (http://
gamapserver.who.int/mapLibrary/). Figure reproduced by permission of the World Health Organization.
S. Melino and M. Paci Progress for dengue virus diseases
FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2987
than one dengue infection during their lifetime.
Sequential heterotypic infection has been shown to

increase virus replication, and thus the probability of
developing DHF, by a process known as antibody-
dependent enhancement (ADE) [5–7]. However, there
are still cases of DHF and DSS that cannot be ade-
quately explained by ADE, an example being in the
confirmed cases of primary infection [8]. Accurate
knowledge of the viral life cycle is essential in order to
highlight potential targets for antiviral therapy and to
obtain key information for the rational design of anti-
viral drugs.
Virus structure and replicative cycle
The structure of the DENv is relatively simple. The
virions are spherical particles 40–50 nm in diameter,
containing three structural proteins: the nucleocapsid
protein C (C; 12–14 kDa); protein M (prM; an 8 kDa
nonglycosylated membrane protein); and the glyco-
protein E (E; 51–59 kDa), which is the major envel-
ope protein present as a homodimer. The DENv
genome is a single-stranded positive-sense RNA that
is encapsidated by protein C in an icosahedral struc-
ture. The genomic RNA presents a single long ORF
encoding the three structural proteins (C, prM and E)
and seven nonstructural (NS1–5) proteins (Fig. 2). It
is translated as a single polyprotein, which is cleaved
by proteases of viral and host origin. Outside the
ORF, there are the 5¢- and 3¢-UTRs, which have sec-
ondary structure and are crucial in the initiation and
regulation of translation, replication and virion
assembly [9–11].
The first step in the viral infection process is the

binding to a cell-surface receptor. This step is mediated
by E protein, identified as a viral attachment protein
for DENv, which leads to virus penetration into the
host cell [12–17]. prM also seems to have an essential
role in the control of the fusion activity of E protein
and is necessary for the correct folding of E protein by
acting as a chaperone-like protein [18–21]. Following
entry and fusion, the translation of the genomic RNA
into a polyprotein is the first event in DENv-infected
cells. A small polypeptide is synthesized, the RNA–
ribosomes–nascent protein complex docks at the endo-
plasmic reticulum (ER), and translation and processing
of the viral polyprotein continue in association with
the ER. Processing of the polyprotein is performed by
cellular and viral proteases. Cleavage at NS1–NS2A
occurs soon after synthesis by a still-unknown host
protease of the ER [22], while cleavages at C–prM,
prM–E, E–NS1 and NS4A–NS4B junctions are per-
formed by a host-cell signal peptidase resident in the
ER, and the cleavages at NS2A–NS2B, NS2B–NS3,
NS3–NS4A and NS4B–NS5 junctions (see Fig. 2) are
performed by the viral serine protease, NS3 [23]. NS
proteins are involved in different functions of the repli-
cative cycle. NS1 glycoprotein (42–50 kDa) is present
on the cell surface. Its colocalization with double-
stranded RNA, together with other evidence, suggests
that intracellular NS1 protein plays a role in the repli-
cation of viral RNA [24,25]. NS2A, NS2B, NS4A and
NS4B are small hydrophobic proteins that are associ-
ated with the membranes. In particular, NS2B is asso-

ciated with the NS3 protease to form an active serine
protease complex [26]. NS3 is implicated in the
C prM E NS1 NS2A NS2B NS3 NS4A NS5 NS4B
Structural proteins Non structural proteins
Furin cleavage
NS2B-NS3 protease cleavage
Signal peptidases cleavage
Unidentified
p
rotease in ER
5’ UTR 3’ UTR
ORF
CAP
Genome organization
Polyprotein and processing
Fig. 2. Organization of the dengue virus
(DENv) RNA genome and scheme of the
proteolytic processing of the DENv poly-
protein.
Progress for dengue virus diseases S. Melino and M. Paci
2988 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS
polyprotein processing and RNA replication. The NS3
protein (69 kDa) is a multifunctional protein with an
N-terminal protease domain (NS3pro) (1–180), an
RNA triphosphatase, an RNA helicase and an RNA-
stimulated NTPase domain in the C-terminal region
[26,27]. The protease and NTPase enzymatic functions
share an overlapping region between residues 160 and
180 of the NS3 protein [28]. The RNA triphosphatase
may contribute to RNA capping [29], whereas the

NTP ⁄ helicase activity may separate nascent RNA
strands from the template [30]. The NS3 viral protease
is absolutely essential (along with the viral-encoded
cofactor NS2B) for viral replication. In addition to the
cleavages at the protein junctions in the polyprotein
described above, the viral serine protease is also
responsible for internal cleavages in the C, NS2A, NS3
and NS4A proteins, the significance of which is not yet
known (see Fig. 2) [31–33].
The most conserved flavivirus protein is NS5. It
is characterized by a methyltransferase motif in the
N-terminal domain and by an RNA-dependent RNA
polymerase located at its C-terminal domain [34,35].
After processing of the viral proteins, most of the NS
proteins associate with the 3¢-UTR of viral RNA to
form a replication complex for RNA synthesis [2]. The
association of protein C with genomic RNA on the
cytosolic face of the ER membrane is the initial step
of virion assembly. The particles are transported
through the secretory pathway to the cell surface for
release.
Dengue vaccine: a possible solution
to DENv infection
In the absence of effective antiviral drugs, vaccination
offers a good chance for decreasing the incidence of
these diseases, live virus vaccines being the most prom-
ising and cost-effective (Table 1). However, currently,
no approved vaccines are available, and various strat-
egies have been used to develop dengue vaccines [36–
38]. Vaccine development has been complicated by the

potential risk of vaccination resulting in the ADE of
future heterotypic infection [36,39]. Different strategies
for the development of dengue vaccines include live
attenuated and inactivated viruses, recombinant sub-
units, protein expression in Escherichia coli, recombin-
ant baculoviruses, recombinant poxviruses, chimeric
viruses derived from infectious cDNA clones of DENv,
and naked DNA vaccines. In preclinical evaluation
using no-human primates, chimeric tetravalent vaccines
have been demonstrated to produce high levels of
neutralizing antibody and viremia protection against
all serotypes after a single dose, and clinical trials are
in progress [37,38,40]. Another type of dengue vaccine
is the DNA vaccine, which represents a promising
gene-based vaccine strategy considered suitable for
developing a dengue tetravalent vaccine [41,42]. Several
flavivirus DNA vaccines, including those against den-
gue, have already been developed [43–46]. Recently, a
new dengue tetravalent DNA vaccine against DENv-3
and DENv-4, based on a prM ⁄ E strategy and com-
bined with two previously constructed DNA vaccines
against DENv-1 and DENv-2, has been constructed
[47]. Molecular biology techniques have facilitated the
development of recombinant subunit vaccines. Several
structural (E and prM) and nonstructural proteins
stimulate immunity, and the nonstructural proteins
NS1 and NS3 are the dominant sources of cross-react-
ive CD4
+
and CD8

+
cytotoxic T-lymphocyte epitopes
[48–51]. Passive immunizations using monoclonal anti-
bodies, and active immunization studies using purified
proteins, provided evidence that these proteins are
important for inducing protective immunity [52–59]. A
synergistic increase in neutralizing antibody titers by
Table 1. Vaccines against dengue fever by the Initiative for Vaccine Research, World Health Organization, updated February 2006.
Type of vaccine
Pharmaceutical
company or
research group
Status of
development
Live attenuated tetravalent vaccine Sanofi-Pasteur ⁄ Mahidol Phase II (pediatric)
Live attenuated 3¢-NCR National Institute of Health Phase I (as monovalent)
Live attenuated two-dose vaccine GlaxoSmithKline PhaseII
Live attenuated DENv-2 and DENv-2 ⁄ 1,2 ⁄ 3,
2 ⁄ 4 chimeric vaccines
Center for Disease
Control and Prevention
Preclinical
Live chimeric virus tetravalent vaccine
dengue ⁄ yellow fever vaccine
Acambis ⁄ Sanofi-Pasteur Phase I (adults)
Tetravalent chimeric, F mutant US Food and Drug Administration Preclinical
Tetravalent E-NS1 fusion protein subunit Hawaii Biotech Preclinical
DNA and recombinant
modified vaccinia Ankara
National Institute of Health Preclinical

S. Melino and M. Paci Progress for dengue virus diseases
FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2989
simultaneous immunization with the DNA and protein
vaccine has been demonstrated [60–62].
Recently, a capsid protein of DEN-2 virus has also
been used in order to obtain statistically significant
protection against the infective homologous virus. This
suggests that effective protection against the four sero-
types might be attainable only by immunization with
the four corresponding capsids, or with one of them
including the immunodominant cytotoxic epitopes of
the others [63].
The major pharmaceutical companies are currently
developing a treatment against the disease. A tetra-
valent live attenuated vaccine was developed at the
Walter Reed Army Institute of Research, Silver
Spring, Maryland, licensed to GlaxoSmithKline [36];
this is the first two-dose vaccine to show a 100%
immune response against all four virus subtypes that
cause the disease. The vaccine is expected to enter
Phase III in 2007 and be commercially available there-
after, if its efficacy and safety is proven.
Therapeutic approaches – NS3 protease
inhibition as a response to DENv
Viral inhibitors have been widely studied in in vitro
systems as supportive medical care and for sympto-
matic treatment; they represent an important aid for
patients and for improving survival in severe forms of
disease. Antiviral therapeutic strategies involve virus-
binding blocking to prevent intracellular virus multipli-

cation and maturation. Based on the putative receptor
role of heparan sulphate for DENv, inhibition of virus
binding and entry has been obtained using polyanionic
compounds such as heparin [64,65], sulphated poly-
saccarides extracted from algae [66,67] or polyoxome-
tallates [68,69], and these have been recently described
as inhibitors of DENv-2 multiplication in Vero cells.
Acetylsalicylate and its metabolite sodium salicylate
specifically inhibit DENv-2 and Japanese encephalitis
virus replication [70]. A specific p38 mitogen-activated
protein kinase inhibitor seems to be involved in the
mechanism of salicylates in suppressing the flavivirus
infection. Recently, in fact, the inhibition of virus rep-
lication through the prevention of virus-associated
apoptosis of infected cells represents a new potential
pharmacological target for the control of flavivirus
infection [71,72]. Inhibitors of viral replication have
also been studied, for example, ribavirin, and inter-
feron-a,-b and -c [73–75]. In recent years, the RNA-
dependent RNA polymerase and the methyltransferase
activity of NS5 protein have been studied as specific
viral targets for chemotherapeutic strategies in order to
prevent RNA strand elongation and RNA capping,
respectively [76]. Recently, nitric oxide has been shown
to suppress DENv RNA and protein accumulation in
infected cells [77,78]. The target of nitric oxide action
in viral RNA synthesis has been investigated and the
selective inhibitory effect on the de novo synthesis
of RNA via the inhibition of RNA-dependent RNA
polymerase activity has been identified [79].

The serine protease domain of NS3 protein plays a
central role in the replicative cycle of DENv [80]. Like
other viral proteases, the DENv NS3 protease repre-
sents an attractive therapeutic target for the develop-
ment of novel antiviral agents. Studies over the past
20 years have shown that many viruses encode one or
more proteases [81,82] that catalyze the processing of
viral polyprotein or maturational processing of precap-
sids and which are required for the production of
infectious virions. The discovery and development of
inhibitors of the viral protease activity assumed clinical
relevance, as has been demonstrated in cases involving
the treatment of patients with acquired immunodefi-
ciency syndrome (AIDS) or hepatitis C virus (HCV)
[83–87]. Studies on the viral protease significantly
increase our understanding of the life cycle of viruses,
the mechanism of proteolytic processing and the regu-
lation of cellular processes. A recurring theme from
structural and sequence analyses is the remarkable
compactness of these enzymes. In addition, most con-
tain no disulfide bridges, in contrast to many classical
cellular proteases, and, moreover, cofactors such as
metal ions or peptides are frequently required to stabil-
ize the viral protease [88–90]. Most viral proteases
have little sequence homology with cellular proteins,
even when they share the same backbone fold. These
characteristics lead to a very different substrate speci-
ficity of the viral proteases with very important impli-
cations for the design and development of their
efficient inhibitors, while undesirable cross reactivity

against cellular enzymes can be minimized.
In the DENv life cycle, proteases from the host (fu-
rin and secretase) and from the virus (NS3 protease)
are required to process the polyprotein precursor into
the individual functional proteins [91], and it has also
been observed that inactivating mutations of the
DENv NS3 protease (NS3pro) cleavage sites in the
polyprotein precursor abolish viral infectivity [92,93].
This suggests that NS3pro is a promising drug target
for flaviviral inhibitors.
Structural and functional studies on NS3
protease
NS3 viral protease is a trypsin-like protease, which,
together with the NS2B cofactor, is essential for the
Progress for dengue virus diseases S. Melino and M. Paci
2990 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS
virus replication. The importance of this protease
activity in viral viability is underscored by the finding
that mutations abolishing the activity, when they are
introduced in the context of an infectious cDNA clone,
eliminate virus recovery [94]. The N-terminal 184
amino acid-long domain of the NS3 multifunctional
protein (69 kDa) is the serine protease (NS3pro) with
a functional catalytic triad (His51, Asp75 and Ser135
in DEN-2). The importance of these catalytic triad
residues in the mechanism of homologous flaviviral
NS3 serine proteases was established by site-directed
mutagenesis of these residues, which abolished pro-
tease activity.
Serine proteases are the best studied of the four clas-

ses (serine, aspartic, metallo and cysteine) of proteases
[95]. The basic mechanism consists of a charge relay
system that transfers the negative charge on the buried
carboxyl via the histidine to the serine. The transfer of
the Ser Oc proton to the histidine converts the serine
into a strong nucleophile for the attack on the peptidyl
carbonyl of the substrate. The substrate is oriented by
the binding of the amino acid side chain of the P
1
resi-
due in the S
1
pocket [96], a hydrogen bond between
the backbone NH of the P
1
residue and two hydrogen
bonds between the carbonyl oxygen of the scissible
bond and two backbone NH groups of the enzyme
(oxyanion binding hole). The reaction is carried on
through a tetrahedral transition state with an acyl-
enzyme intermediate.
The DENv NS3 protease is also commonly desig-
nated as being a member of the flavivirin enzyme
family (EC 3.4.21.91 and S07.001 Peptidase MEROPS
peptidase database ), which
comprise the NS2B–NS3 endoproteases of the Flavivi-
rus genus [97,98]. The presence of a small activating
cofactor protein is a prerequisite for the optimal cata-
lytic activity of the flaviviral proteases with natural
polyprotein substrates [99,100]. The DENv NS3 pro-

tease requires the presence of the nonstructural NS2B
protein for its activity [26]. The NS2B–NS3 conjugate
has been shown to cleave the precursor polypotein
at NS2A ⁄ NS2B, NS2B ⁄ NS3, NS3 ⁄ NS4A and
NS4B ⁄ NS5 junctions, as well as at internal sites
within C, NS2A, NS3 and NS4A [23,101,102]. The
NS2B protein is composed of seven domains, which
can be separated on the basis of their relative hydro-
phobicity (domains I–VII) [103]. The hydrophobic
core residues, belonging to domain IV (G69–E80),
were proposed to interact with NS3pro. This domain
is flanked by two hydrophilic stretches (domains III
and V). Studies using mutant plasmids transfected
into cells have shown that the fragment of 40 residues
of the NS2B encompassing domains III to V, is the
minimal region necessary for inducing the protease
activity of NS3pro [104,105]. Moreover, the NS2B–
NS3 association, demonstrated by co-immunoprecipi-
tation experiments, is also mediated by this hydrophi-
lic region [104]. Comparing the kinetic properties of
NS3 and NS2B–NS3, it has been suggested that
NS2B generates additional specific interactions with
the P2 and P3 residues of the substrates [106]. Cur-
rently, the molecular details of the mechanism by
which the NS2B cofactor stimulates the activity of
the protease are not yet known. The analogy with the
HCV protease has offered some structural and mech-
anistic explanations for the activation of this flaviviral
protease by its cofactor. However, unlike the HCV
NS3 protein analog, it seems that in the case of the

DEN-2 NS3pro the cofactor activity cannot be sup-
plied in trans with a small peptide derived from the
cofactor NS2B [105]. Other serine proteases (subtil-
isin, a-lytic protease) are also known to require a
pro-region, such as NS2B, for inducing a productive
folding leading to the active form. In these cases,
once the protein is folded, the necessary pro-region
does not remain bound to the active enzyme. The
results obtained regarding the NS2B–NS3pro complex
indicate that NS2B also functions as a molecular
chaperone in assisting the folding of NS3pro to the
active conformation [105,107]. A new construct of the
recombinant form of the NS3pro fused to a 40-resi-
due cofactor and corresponding to the hydrophilic
part of NS2B by a glycine linker was engineered and
expressed in E. coli, and demonstrated activity against
hexapeptide substrates modified as chromogenic para-
nitroanilide derivates [105]. Expression of the con-
struct CF40GlyNS3pro (the amino acid sequence is
shown in Fig. 3) resulted in substantially high yields
of the soluble and active recombinant protein, which
was significantly more active than the refolded
NS3pro and CF40NS3pro (lacking the Gly linker). In
fact, although the DENv NS3 protease exhibits
NS2B-independent activity with small substrates such
as N-a-benzoyl-l-arginine-p-nitroanilide, the activity
towards peptide substrates is stimulated significantly
in the presence of the NS2B protein [26,106].
Recently, it has been proposed that the Fx
3

F motif
is the common structural element involved in cofactor
binding to the protease [108]. This motif consists of
two bulky hydrophobic residues separated by three
unspecified residues; it has been speculated that addi-
tional residues, located outside this sequence motif,
would contribute to the stringent specificity of the pro-
tease for the corresponding polyprotein substrate [108].
A mutagenesis study with the DENv NS2B cofactor
has revealed that substitution of the F residues (corres-
S. Melino and M. Paci Progress for dengue virus diseases
FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2991
ponding to residues Leu75 and Ile79) with alanine
results in a decrease of the NS2B–NS3pro autoprocess-
ing to approximately 55 and 75% of the wild-type
value [109]. By contrast, the replacement of W61
located outside this sequential motif yielded a catalyti-
cally inactive enzyme [109]. Moreover, in agreement
with these results, the W61 residue is also present in
the CFNS3d protein complex, which is an active form
of the enzyme obtained by limited proteolysis of the
CF40GlyNS3pro [107]. All these results suggest a
pivotal function for this invariant residue in protease
activation.
Other experiments, examining the role of NS2B
cofactors, indicate that in addition to activating pro-
teolytic activity, NS2B is necessary for promoting
membrane association of the NS3 complex [110], and
cryoimmunoelectron microscopy studies have sugges-
ted that functional NS2B–NS3 proteolytic activity may

be compartmentalized into specific membranous struc-
tures [111]. This finding suggests that the protease
activity may be affected by the membrane environ-
ment; in fact, the CF40GlyNS3pro activity in vitro was
increased by the presence of zwitterionic and nonionic
detergents at low concentrations [105].
Structural biological studies
The initial structural study was performed by Brink-
worth et al. [103], using a sequence homology
approach of NS3 protease with HCV NS3 protease,
which has been widely studied and whose structure
has been resolved by X-ray and NMR spectroscopy
[112–114]. By molecular modelling, a number of
insights concerning the cofactor interaction and sub-
strate specificity were obtained.
The model, by analogy with HCV NS4A, predicted
that the NS2B peptide encompassing residues Gly72–
Gly83 could be sufficient to function as a peptide
cofactor in vitro [103]. Moreover, the model suggested
a substrate specificity in the P1 position for the basic
Lys or Arg residues because of the presence of an aci-
dic Asp129 residue, present in the active cleft at six
residues before the catalytic Ser135 and conserved in
all the flavivirus sequences. Other interactions between
DEN2 NS3pro and the substrate have been predicted
by this model, such as a possible H-bond between the
Asp75 and the P2 residues and a hydrophobic interac-
tion between the P1¢ residue and the Val52 or Tyr41
residues. The resolution of the crystal structure of
NS3pro [115] at 2.1 A

˚
has been reported (Fig. 4A).
This structure differs significantly from that of HCV
NS3pro, resembling more that of HCV NS3pro in
complex with the cofactor NS4A and, in particular,
the first tract of 30 residues of the protein appears with
a different conformation. In particular, the structure
obtained shows a rather limited extension cleft region
between the two domains of DEN-2 NS3pro and it
was not useful in elucidating the specific interactions
with the substrate beyond P2 and P2¢.
For these reasons, the structure obtained in the
absence of the cofactor could not be used to design
specific inhibitors, although it represented an import-
ant starting point for the determination of the DEN-2
NS2B–NS3pro complex structure. The X-ray structure
of the complex NS3pro with a Bowman Birk inhibitor
[116] has been compared with the results reported for
the DEN-2 NS3pro.
Differences are particularly pronounced for the
stretch of residues 127–136, including the catalytic
Ser135 residue, and lead to a different orientation
of Asp129, which has electrostatic interactions with
the P1 Arg residue. However, these observations
may not have physiological implications considering
that the regulatory component, NS2B, was not present
in the complex. The structural NMR studies on
CF40GlyNS3pro show the presence of a substantially
flexible or unfolded region of the protein that is
responsible for the aggregation at high concentrations

and makes the determination of the solution structure
very unlikely [107].
Fig. 3. Sequence alignment between DENv-2
NS2B–NS3pro (2FOM pdb) and West Nile
virus NS2B–NS3pro (2FP7 pdb) obtained
using the
T-COFFEE program, version 1.41
( />tcoffee_cgi/index.cgi) [135]. The catalytic
residues are in bold and the numbers refer
to the DENv-2 NS2B–NS3pro sequence.
Progress for dengue virus diseases S. Melino and M. Paci
2992 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS
Recently, the crystallographic structure (at 1.5 A
˚
resolution) of the active form of the NS2B–NS3pro
protein, including the 47-residue core region of NS2B
via a glycine linker (such as CF40GlyNS3pro), has
become available (2FOM pdb; Fig. 4B) [117]. Overall,
the structure is topologically close to that reported pre-
viously (six b-strands in two b-barrels with the cata-
lytic triad located at the cleft between the two barrels).
Nevertheless, it presents relevant differences in the sec-
ondary and tertiary structure that are important for
definition of the structural and functional roles of the
NS2B cofactor. However, the X-ray structure does not
appear to be structurally well defined in some regions.
This suggests that these regions may adopt multiple
conformations when passing from the solution to the
crystal state, as has also been observed in the NMR
experiments.

Differences have also been found in the length and
location of secondary structural elements, which
assume great importance for the solubility and proteo-
lytic activity of this protein form. As observed for the
complex of HCV NS4A–NS3pro [112,113], NS2B con-
tributes a b-strand (residues 51–57) to the stability of
the N-terminal b-barrel of NS3 in contrast to the pre-
viously reported prediction by homology modelling,
where the interacting fragment of the NS2B was the
70–81 tract [103].
On the other hand, the expression of a truncated
NS2B–NS3pro form, including only the 40–66 residues
of NS2B, gives a soluble but catalytically inactive form
of the enzyme [117]. This suggests that the region 40–
66 of the cofactor is important in the folding of the
protein and that the C-terminal part of the cofactor,
which is absent in the truncated form, directly interacts
with the substrate-binding site. On the other hand,
the soluble form of the CFNS3d protein complex,
obtained by limited proteolysis of CF40Gly–NS3pro,
which conserves 52% of the proteolytic activity, con-
sists of the D6–E179 region of NS3pro and the NS2B
fragment D50–E80. The
1
H-
15
N heteronuclear single
quantum coherence spectrum of the uniformly labelled
15
N-CFNS3d shows a good cross-peak dispersion,

indicating a stable folded state of the protein [107]. All
data confirm that the NS2B fragment D50–E80 has a
strong interaction with NS3pro and is also able to pro-
mote in trans the activity of the enzyme when correctly
folded. This finding indicates that this cofactor region
has an important role in the conformational stability
of the active site. In the crystal structure, the electron
density beyond the NS2B residue 76 is discontinuous,
revealing that this region may adopt several conforma-
tions probably as a consequence of its great flexibility
in solution. No evidence of direct interactions of NS2B
with the active site are found in this structure, giving
no structural explanation of its absolute requirement
by NS3pro for activity.
On the contrary, the crystallographic structure of
the homologous NS2B–NS3pro protein of West Nile
virus has shown direct interactions of the C-terminal
part of NS2B with the active site of the NS3pro. The
C-terminal part of NS2B wraps around NS3pro and,
in particular, the Arg78–Leu87 residues form a
b-strand in NS2B, which links the N-terminal tract of
NS3pro. The structure of the West Nile virus NS2B–
NS3 pro-inhibitor complex has also elucidated (2FP7
pdb) [117], the details of the S1 pocket, formed by
Gly151, Tyr161, Tyr150, Asp129 and the backbone
C-Term
Asp 75
A
N-Term
His 51

Ser 135
N-Term
C-Term
B
NS2B 43-96
Fig. 4. Ribbon representations of the NS3 protease (NS3pro) (1BEF
pdb) (A) and the NS2B–NS3pro (2FOM pdb) (B) structures. The
NS2B 43–96 fragment [108,110] is shown in blue. In red are the
side chains of the catalytic residues His51, Asp75 and Ser135.
S. Melino and M. Paci Progress for dengue virus diseases
FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2993
residues of Tyr130–Thr132. Asp129 is located at the
bottom of the pocket, stabilizing the positively charged
side chain of P1 arginine. It has also been reported
that in this case the S2 pocket is dominated by the
negative electrostatic charge originating from the
NS2B residues Asp82, Gly83 and Asn84 that are close
to the positively charged guanidinium group of the P2
arginine [117]. These findings could also explain the
importance of a basic residue at P2 for the DEN-2
NS2B–NS3pro protein, and are in agreement with the
observed loss of binding when the P2 arginine is
replaced by an alanine residue. The importance of this
contribution is in the observation that the formation
of the active protease differs substantially from those
observed with other cofactor-activated viral proteases,
such as HCV NS4A–NS3pro [113,114]. The structures
currently available explain well the huge increase in
activity of flaviviral NS3pro in the presence of NS2B,
and may be useful in the development of drugs to treat

the flaviviral diseases.
Substrate specificity of NS2B–NS3pro
The first step towards designing an inhibitor of the
viral protease is to identify substrate specificity. The
selectivity of the proteases for particular substrates
results from the presence of specific binding sites
on the enzyme for amino acid side chains of the
substrate(s). The virus-encoded proteases display an
unusual degree of selectivity for their natural
polyprotein substrates and only very few cases are
known where the viral enzyme reacts with protein
substrates derived from the host cell [118,119]. In the
case of viral proteases, the identification of a high
turnover substrate is usually difficult [120] because
the kinetic parameters of synthetic peptides based on
the natural cleavage sites are generally unfavorable
[121].
The NS3 protease in the absence of the cofactor
reacts with small model substrates for serine proteases,
such as N-a-benzoyl-l-arginine-p-nitroanilide, and acti-
vity of the NS3 protease towards the substrate is
higher than that of the NS2–NS3 complex [106]. This
suggests that substrate recognition in the complex
requires additional interactions, extending beyond the
P1 site, for optimal activity. Other studies have indica-
ted that NS2B–NS3pro requires the presence of
Lys ⁄ Arg and Arg, respectively, at the P2 and P1 posi-
tions, for achieving substrate proteolysis, and that the
cleavage motifs have features in common with the phy-
siological cleavage sites [122,123]. The best substrate

identified, using synthetic combinatorial libraries of
peptides and single substrate kinetics, is the fluorogenic
peptide Bz-nKRR-acmc, which shows, for DEN-2 pro-
tease, an apparent K
m
value of 12 ± 2 lm,ak
cat
of
1.4 ± 0.1Æs
)1
and a catalytic efficiency, expressed as
k
cat
⁄ K
m
, of 112 100 ± 18 500Æm
)1
Æs
)1
[122]. The sub-
strate pocket of the NS3 proteases from the four sero-
types consists of a number of highly conserved
residues within the S1–S4 region. The enzymes of the
four serotypes appear to share very similar substrate
specificities, which implies that it is possible to develop
a single inhibitory agent targeting all four dengue
NS3 proteases [122]. Basic or aliphatic residues
at P3 and P4, and small or polar residues at P1¢
(Ser > Gly > Ala), are required [123,124]. Moreover,
the P3 and P4 positions also contribute significantly to

ground state binding, providing additional evidence for
enzyme–substrate interactions that extend beyond S2
to S2¢ [122]. The introduction of an arginine residue at
P3 results in an almost four-fold increase in k
cat
⁄ K
m
,
and the introduction of an arginine residue at P3 and
P4 in the capsid protein-derived tetrabasic sequence
RRRR results in a 30-fold increase in k
cat
⁄ K
m
.
A higher degree of selectivity for serine at the P3¢ posi-
tion is needed, whereas selection of residues at the P2¢,
and especially at the P4¢ positions seems to be relat-
ively unrestrained [123]. Recently, the specifics of sub-
strate recognition by NS3pro from DENv have been
mapped using a library of the 9-mer peptides to the
cleavable sequences with the general P4–P3–P2–P1–
P1¢–P2¢–P3¢–P4¢–Gly structure [124]. The N terminus
and the constant C-terminal Gly of the peptides were
tagged with a fluorescent tag and with a biotin tag,
respectively. The amino acid sequences of the peptides
corresponding to the junction regions efficiently clea-
ved by the DENv protease are shown in Table 2. In
addition, other potential sites of the NS2B–NS3pro
that are efficiently cleaved have been identified, also on

the basis of the high homology with the West Nile
virus NS3 protease [124]. These sites are in the
NS3pro ⁄ helicase protein and correspond to the
sequences 1659RKKRRLTIM1666, 1674KTKRYLP-
A1681 and 1930AQRRGRIG1937 [117]. A library
obtained by randomization of the P1¢ and P2¢ posi-
tions of the peptide 2522GKRGGAK2529 with differ-
ent amino acids has been used to demonstrate that the
NS2B–NS3pro can accommodate, in these positions, a
number of the amino acid residues, including the bulky
hydrophobic Trp, Phe and Tyr, but does not tolerate
the presence of the negatively charged Asp and Glu
residues [124]. In contrast, the homologous proteases
from West Nile virus and from Japanese encephalitis
virus prefer small (like Gly) or polar amino acid resi-
dues in the P1¢ and P2¢ positions. Noteworthy, the
West Nile virus protease processes substrates with a P2
Progress for dengue virus diseases S. Melino and M. Paci
2994 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS
Lys more efficiently than those with a P2 Arg, in con-
trast to the four serotypes of dengue protease [122],
which are more active against substrates with Arg
instead of Lys at P2. Recent work shows that the co-
factor residue at NS2B-84 is associated with a prefer-
ence for Lys or Arg at the substrate P2 position. In
particular, the presence of an Asn or an Asp residue
at NS2B-84 leads to a preference for a Lys residue at
P2 of the native substrate, while the substitution with
Ser, Thr or Glu at NS2B-84 leads to a preference for
an Arg residue at the P2 position [125]. Thus, the

finding that DENv proteases exhibit a preference for
Arg at the P2 position could be explained by the
presence of Ser or Thr at NS2B-84 [125]. On the
basis of these recent studies, the DENv enzyme seems
to adopt a restricted specificity to process the natural
cleavage sites of the polyprotein precursor, but this
specificity is less stringent than the homologous viral
proteases.
Inhibition of NS3 protease, a therapeutic target
In a first step towards design of an inhibitor for the
DENv NS3 serine protease, the standard inhibitors of
serine proteases have been assayed. The serine protease
inhibitor, aprotinin, has been shown to inhibit the four
CF40GlyNS3pro proteases with high affinity (K
i
¼ 79,
25, 88, 6.4 pm for DEN 1–4 CF40GlyNS3pro pro-
teases, respectively), whereas other serine protease
inhibitors show a low ability in inhibiting the viral
protease [105,122]. Similarly to the HCV NS3 protease,
the existence of a high-affinity binding site in the non-
prime region of the enzyme offers the possibility of
developing effective inhibitors against the DENv pro-
tease by combinatorial optimization of the cleavage
sites. For this reason, small-molecule inhibitors based
upon the peptide substrates have been synthesized as
inhibitors of NS3pro (Table 3). N-terminal cleavage
site peptides, corresponding to the P6–P1 region of the
Table 3. Representative competitive inhibitors of the dengue NS2B–NS3pro serine protease.
Type of compound Compound K

i
value (lM) Reference
a-keto-amide Ac-FAAGRR-a keto–SL-CONH
2
47 [105]
Amide Ac-FAAGRR-CONH
2
25.87 [126]
Ac-RTSKKR-CONH
2
12.14 [126]
Ac-KKR-CONH
2
22.31 [126]
Aldehyde Ac-FAAGRR-CHO 16 [105]
Bz-Nle-Lys-Arg-Arg-H 5.8 [131]
Bz-Ala-Lys-Arg-Arg-H 5.3 [131]
Bz-Phe-Lys-Arg-Arg-H 6.8 [131]
Bz-Nle-Lys-Arg-Phe-H 15.9 [131]
Bz-Nle-Lys-Arg-Trp-H 7.5 [131]
Bz-Nle-Phe-Arg-Arg-H 15.8 [131]
Bz-
D-Nle-Lys-Arg-Arg-H 9.4 [131]
Bz-Lys-Arg-Arg-H 1.5 [131]
Bz-Arg-Arg-H 12.0 [131]
Bz-Nle-Lys-Arg-Arg-(p-guanidinyl)Phe-H 2.8 [131]
Trifluoromethyl ketone Bz-Nle-Lys-Arg-Arg-CF
3
0.85 [130]
Boronic acid Bz-Nle-Lys-Arg-Arg-B(OH)

2
0.043 [130]
Cyclohexenyl chalcone
derivative
Panduratin A 25 [133]
4-Hydroxypanduratin A 21 [133]
Table 2. The amino acid sequences of the cleavage sites of the NS2B–NS3pro protease in the precursor polyprotein.
fl
Denotes the scissile
bond, and the P1 residues are shown in bold.
S. Melino and M. Paci Progress for dengue virus diseases
FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2995
polyprotein, were found to act as competitive inhibi-
tors of the enzyme, with K
i
values ranging from 67 to
12 lm. The NS2A ⁄ NS2B cleavage site, RTSKKR, is
the peptide with the lowest K
i
value. However, in con-
trast to HCV NS3 protease, the cleavage products and
their analogs do not appreciably inhibit this protease.
In fact, the peptides corresponding to the P1¢–P5¢
region of the polyprotein cleavage sites do not show
any inhibitory effect on enzymatic activity, even at
1mm concentration [126].
Peptidic a-keto amide inhibitors have been well char-
acterized as reversible competitive inhibitors for other
serine proteases, including HCV NS3 protease [127–
129]. Similarly, in the case of DENv NS3 protease, some

peptidic a-ketoamide inhibitors have been synthesized
[105,130] in order to determine their inhibitory efficacy.
Except for the a-ketoamide peptide Ac-FAAGRR-a-
keto-SL-CONH
2
[105], which showed a K
i
value of
47 lm, the other synthesized a-ketoamide peptides
showed no effect up to 500 lm concentration [130].
Substrate-based peptide aldehydes have also been
synthesized, resulting in low micromolar inhibitors
with a K
i
value up to 1.5 lm (see Table 2) [105,131]. A
systematic structure–activity relationship study has
revealed that the P2 Arg residue is more important for
enzyme interactions than P1 Arg and that the dipep-
tide aldehyde inhibitors have a low micromolar activity
[131]. Furthermore, the replacement of P1 arginine
with Phe and Trp does not induce a loss of the inhi-
bitory activity [131]. In the published structure of
DEN-2 NS3pro-MbBBI (1DF9), obtained in the
absence of the NS2B cofactor, the P1 Arg residue has
electrostatic interactions with Y150 and S163 and
forms two H-bonding interactions with D129
[116,132]. Thus, when P1 Arg is replaced with Phe or
Trp, the hydrogen bonds with Y150 and S163 are lost,
but p–p interactions between P1 Phe ⁄ Trp and Y150
are possible and stabilize inhibitor binding [131].

Potent inhibitors have been identified by incorporating
trifluoromethyl ketone and boronic acid onto the sub-
strate peptide [130]. Tetrapeptide boronic acid proved
to be the most potent inhibitor of DENv NS3 prote-
ase, having a K
i
of 43 nm, whereas the trifluoromethyl
ketone occupies an intermediate position between that
of peptide aldehyde and peptide boronic acid [131].
Recent studies have shown that some natural com-
pounds, such as chalcones isolated from Boesenbergia
rotunda (a common spice belonging to a member of
the ginger family), are able to inhibit the DENv NS3
protease. In particular, the cyclohexenyl chalcone deri-
vates, 4-hydroxypanduratin A and panduratin A, show
good competitive inhibitory activities against DEN-2
virus NS3 protease, having apparent K
i
values of 21
and 25 lm, respectively [133]. Although several inhibi-
tors of DENv proteases have been tested, selective
viral protease inhibition has not been obtained to date
and inhibitors for clinical trials are not yet available.
Conclusions
Albeit there are still no specific vaccines or chemother-
apy regimes for the prevention and treatment of DF
and DHF, the understanding and the biochemical
characterization of the life cycle of DENv have made
substantial progress over the past few years, and all
the life cycle stages represent potential targets for anti-

viral drug discovery.
The DENv NS3 protein, as in other viral patholo-
gies, is a valid molecular target for the development of
antiviral compounds. Inhibitors against the DENv pro-
tease will be of great benefit in the design of drugs to
combat other related flaviviruses, as well as Japanese
encephalitis virus and West Nile virus.
In vitro inhibitors against NS2B–NS3pro protease
for the four serotypes are now available. However, the
fact that the protease activity in vitro of the NS2B–
NS3pro (or CF40GlyNS3) is present only at alkaline
pH, suggests that in the physiological environment of
the host cell, further interactions with an unknown
activator or post-translational modifications are
required for optimal activity. This situation could be
similar to that of kallikrein. Recently, in fact, glycoso-
aminoglycans or kosmotropic salts have been found to
induce a relevant increase of human kallikrein 6 acti-
vity [134]. In the presence of these compounds, the
optimum pH of this secreted serine-type protease shif-
ted towards lower values (from pH 9 towards pH 7.5)
[134].
The interaction between the NS2B cofactor and
NS3pro seems to be important not only for the correct
fold of the protease but also for the correct interaction
with the substrate. The binding site of the NS2B cofac-
tor could be examined for a view to inhibitor develop-
ment, and competitive inhibitors of the binding site
might be able to inhibit the correct fold of the protease.
At present, no inhibitors of the cofactor–protease bind-

ing are available, although the resolution of the crystal-
lographic structure and the production of mutants can
help to develop specific inhibitors of the binding to the
cofactor. Some regions of the NS2B–NS3pro structure
are not yet well defined, and their resolution will be
important for the complete understanding of the struc-
ture–function correlations, such as the resolution of the
structure of its complex with an inhibitor.
On the other hand, the NTPase ⁄ helicase region of
the NS3 protein, and the surface of NS3 protein with
Progress for dengue virus diseases S. Melino and M. Paci
2996 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS
the NS5 replicase, could represent alternative drug tar-
gets. Thus, the use of a pharmacological therapy using
combinations of different inhibitors, similarly to other
viral therapies, could minimize the development of
rapid resistance.
In conclusion, considerable effort has recently been
made towards inhibiting the viral replication of DENv.
Much remains to be performed to achieve results suit-
able for experimentation in clinical trials and to pro-
duce a drug for blocking the DENv spread. To date,
funding for a co-ordinated strategy against dengue has
been disappointing. This is probably attributable to
the spread of dengue diseases in the world’s poorest
countries. Our hope therefore is that the major inter-
national funding agencies will now seriously consider
increasing their commitment to combat these diseases.
The need is all the more urgent now that climate
change has made the spread of DENv to Western

countries a real possibility. Further research therefore
is in the future interest of the people of both rich and
poor nations alike.
Acknowledgements
The authors would like to thank Renato Sabelli for his
continuous work in the field during the elaboration of
this manuscript. The work was partly supported by
grants PRIN and FIRB of Italian MIUR and by the
Ministry of External Affaire of Italy.
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