Inhibition of the NS2B-NS3 Protease – Towards a

Causative Therapy for Dengue Virus Diseases

Gerd Katzenmeier#

Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University,
Salaya Campus, Phutthamonthon No. 4 Rd., Nakornpathom 73170, Thailand

Abstract
The high impact of diseases caused by dengue viruses on global health is now reflected in an increased
interest in the identification of drug targets and the rationale-based development of antiviral inhibitors
which are suitable for a causative treatment of severe forms of dengue virus infections – dengue
haemorrhagic fever and dengue shock syndrome. A promising target for the design of specific inhibitors is
the dengue virus NS3 serine protease which – in the complex with the small activator protein NS2B –
catalyses processing of the viral polyprotein at a number of sites in the nonstructural region. The NS3
protease is an indispensable component of the viral replication machinery and inhibition of this protein
offers the prospect of eventually preventing dengue viruses from replication and maturation. After nearly
a decade of mainly genetic analysis of flaviviral replication, recent studies have contributed substantial
biochemical information on polyprotein processing including the 3-dimensional structure of the dengue
virus NS3 protease domain, the mechanism of co-factor-dependent activation and sensitive in vitro
assays which are needed for studies on substrate specificity and the development of high-throughput
assays for inhibitor screening. This review discusses recent biochemical findings which are relevant to the
design of potential inhibitors directed against the dengue virus NS3 protease.
Keywords: Dengue virus, NS2B/NS3, polyprotein, protease, inhibitor, treatment.

Viral polyprotein processing nucleotides and encodes a single

Dengue viruses, members of the Flaviviridae
family, possess single-stranded, positive
sense RNA genomes and generate mature
viral proteins by co- and post-translational
proteolytic processing of a polyprotein
precursor catalysed by host cell and virus-
encoded proteases [for review see refs. 1, 2
and references herein]. The genomic RNA
of dengue virus serotype 2 contains 10,723
polyprotein precursor of 3,391 amino acid
residues[3] . Individual viral proteins are
arranged in the order C-prM-E-NS1-NS2A-
NS2B-NS3-NS4A-NS4B-NS5. Proteolytic
cleavages in the N-terminal region of the
viral polyprotein are mediated by a host
signal peptidase and yields three structural
proteins C, prM and E, which constitute the
virion[4] . Before the virion exits the cell, prM
is cleaved by a cellular furin-type

#
E-mail: frkgz@mahidol.ac.th; Tel.: (662)-800-3624-1237; Fax: (662)-441-9906

58 Dengue Bulletin – Vol 28, 2004


prohormone convertase in the post-Golgi
acidic compartment to yield the M protein[5] .
Cleavages at the NS1/NS2A and
NS4A/NS4B junctions are catalysed by a
signalase bound to the membranes of the
ER [6,7] . Proteolytic cleavages in the
nonstructural region of the polyprotein are
mediated by a heterodimeric complex of
NS3 with the activator protein NS2B which
catalyses in cis (intramolecular) cleavages at
the NS2A/NS2B and NS2B/NS3 sites and in
trans (intermolecular) cleavages at the
NS3/NS4A and NS4B/NS5 polyprotein
junctions[8-10] . Additional cleavages
mediated by the NS3 protease within the C,
NS2A, NS4A and within a conserved C-
terminal portion of NS3 itself have been
described in the literature [11-13] .
Figure 1. The 3-dimensional structure of
the dengue virus NS3 protease
(shown here is a superimposition of a
ribbon-presentation of the Cα trace on a
space-filling surface model. Residues of
the catalytic triad (His51, Asp75, Ser135)
are shown as sticks. The figure was
generated by Deepview Swiss-Pdb Viewer)
Ser135
His51
Asp75
Dengue Bulletin – Vol 28, 2004
Inhibition of NS2B/NS3 Protease
Cleavage sites recognized by the
NS2B/NS3 protease consist of ‘dibasic’
residues Lys-Arg, Arg-Arg and Arg-Lys at the
(nonprime) P1 and P2 positions of the
cleavage site sequence followed by short
chain residues such as Gly, Ala and Ser at
the (prime) P1’ position. The “non-
canonical” NS2B/NS3 site contains a Gln
residue at the P2 position (Figure 1).
The NS3 protease domain
The existence of a trypsin-like serine
protease domain in the N-terminal region of
the flaviviral NS3 proteins was originally
predicted by sequence comparisons between
cellular and virus-encoded proteases[14]. The
NS2B-NS3 endopeptidases of the Flavivirus
genus which at present comprises at least 68
known members, are now commonly
designated as flavivirin (EC 3.4.21.91) [15,16] .
The dengue virus 69 kDa NS3 protein is a
multifunctional protein with a serine
protease domain located within the N-
terminal 167 amino acid residues[17] and
activities of a nucleoside triphosphatase
(NTPase) and RNA helicase in the C-
terminal moiety[18] . A catalytic triad
consisting of residues His51, Asp75 and
Ser135 was identified by site-directed
mutagenesis experiments and replacement
of the catalytic serine by alanine resulted in
an enzymatically inactive NS3 protein[19] .
The NS3 protease is an essential component
for maturation of the virus and viable virus
was never recovered from infectious cDNA
clones carrying mutations in the NS3
sequence which abolished protease
activity[20] . Interaction of the helicase
portion of NS3 with the viral RNA-
dependent RNA polymerase NS5 may
promote the association of the viral
replicase complex to the membranes of the
ER [21] .
59
Inhibition of NS2B/NS3 Protease
Figure 2. Schematic of dengue virus polyprotein processing
(shown here are sites on the flavivirus polyprotein cleaved by host-encoded proteases and
the virus-encoded two-component protease NS2B-NS3)
C prM E NS1 NS2A
NS2BNS3 NS4A
The 3-dimensional structure of the N-
terminal 185 residues of the dengue virus
NS3 protease domain (NS3pro) was
resolved at a resolution of 2.1 Å[22] . The
overall folding of the protein resembles the
6-stranded β-barrel conformation typical for
chymotrypsin-like serine proteases.
Interestingly, the structure of the dengue
virus NS3 protease is closer to that of the
hepatitis C virus NS3/NS4A co-complex
than to the unliganded HCV NS3 protease,
an observation which is suggestive of major
structural differences in the co-factor-
dependent activation mechanism of the two
proteases[23] . The substrate binding site of
NS3pro is relatively shallow and contourless
and specific enzyme-substrate interactions
were not predicted to extend beyond the
P2 and P2’ positions of the substrate
peptide in the absence of the NS2B co-
factor[22] (Figure 2).
The NS2B co-factor
The presence of a small activating protein or
co-factor is a prerequisite for optimal activity
of the flaviviral NS3 proteases with their
60
NS4BNS5
NS2B/NS3 protease
Signal peptidases
Furin protease
Unidentified protease
natural polyprotein substrates. Although the
dengue virus NS3 protease exhibits NS2B-
independent activity with model substrates
for serine proteases, enzymatic cleavage of
dibasic peptides is markedly enhanced with
the NS2B-NS3 co-complex and the
presence of the NS2B activation sequence is
indispensable for the cleavage of
polyprotein substrates in vitro[24] . The initial
characterization of the co-factor
requirement for the dengue virus NS3
protease had revealed that the minimal
region necessary for protease activation was
located in a 40-residue hydrophilic segment
of NS2B[25] . The hydrophobic flanking
regions of the 14 kDa NS2B protein are
likely to be involved in targeting the
protease complex to the membranes of the
ER where genome replication occurs. Fusion
of the NS2B core sequence to the NS3
protease domain yielded a catalytically
active NS2B(H)-NS3p protein, which, upon
expression in E. coli and subsequent
refolding, displayed autoproteolytic
processing at the NS2B/NS3 site conducive
to the formation of a non-covalent adduct[24] .
Incorporation of a flexible nonamer linker,
Gly4 -Ser-Gly4, between the NS2B core
Dengue Bulletin – Vol 28, 2004

segment and the protease domain resulted
in a cleavage-resistent protease with
optimized enzymatic activity against
hexapeptide substrates representing native
polyprotein cleavage junctions[26] . A
recombinant construct representing the full-
length NS2B co-factor linked to the NS3
protease domain was enzymatically active
with peptide substrates derived from the
polyprotein; however, this protein was
completely resistant to proteolytic self-
cleavage[27,28] .
An essential requirement for the correct
association of the co-factor with the
protease is the presence of hydrophobic
residues which act as ‘anchor’ for the
protease – co-factor interaction. Recently,
based on mutagenesis experiments and
sequence comparisons of known flaviviral
co-factors, the “Φx3Φ” - motif was proposed
as the common structural element involved
in co-factor binding to the protease[29] . The
“Φx3Φ” - motif is comprised of two bulky
hydrophobic residues separated by three
unspecified residues and it was
hypothesized that additional residues
located at the N-terminus of the activation
sequence would contribute to the stringent
specificity of the protease for the
polyprotein substrate. A mutagenesis study
with the dengue virus NS2B co-factor had
revealed that substitutions of the “Φ” -
residues (Leu75 and Ile79 in NS2B of
dengue virus type 2) by alanine resulted in
preponderant effects on the catalytic activity
of the NS3 protease rather than on substrate
binding [P. Niyomrattanakit, unpublished
data]. A single residue in the N-terminal
region of NS2B, Trp62, was critical for
protease activation and replacement of this
residue yielded a NS3 protease which was
catalytically inactive in autoproteolysis and
Dengue Bulletin – Vol 28, 2004
Inhibition of NS2B/NS3 Protease
reaction with the synthetic substrate peptide
GRR-AMC.
For the HCV NS3-NS4A protease
complex, large structural rearrangements
leading to a catalytic triad, which is
conformationally optimized for proton
shuttle during catalysis, were observed as a
result of co-factor binding[30] . No 3-
dimensional structure is available for the
dengue virus NS2B-NS3 co-complex and
the precise mechanism of co-factor-
dependent activation is not fully elucidated
as yet. In particular, it is an open question as
to whether binding of the substrate
contributes to the formation of an ‘induced
fit’ conformation as observed with the HCV
NS3 protease[31,32] .
Substrate specificity
So far, only very limited efforts have been
undertaken to analyse the precise substrate
requirements and determinants of cleavage
efficiency for the dengue virus NS3 protease.
This is surprising in the light of the fact that
development of inhibitors against serine
proteases usually starts with optimal peptide
substrates derived from the nonprime side
wherein the scissile amide bond is replaced
by an electrophile which reacts with the
catalytic serine residue.
The NS3 protease reacts with small
model substrates for serine proteases such as
N-α-benzoyl-L-arginine-p-nitroanilide (BAPA)
and activity of the unliganded NS3 protease
towards this substrate is higher than that of
the NS2B-NS3 co-complex[24] , a finding
which suggests that substrate recognition in
the complex requires additional interactions
extending beyond the P1 side for optimal
activity. A number of fluorogenic tripeptides
containing dibasic residues at the P1 and P2
61
Inhibition of NS2B/NS3 Protease
positions are cleaved by NS2B(H)-NS3pro
protease and the best substrate identified in
these experiments was GRR-AMC, which
had a Km value of 180 µM, a kcat value of
0.031 s-1 and a catalytic efficiency expressed
as kcat / Km of 172 s-1 M-1 [24] .
Chromogenic p-nitroanilide peptides
representing hexameric sequences of the
native polyprotein cleavage junctions were
tested in a photometric assay and the most
efficiently cleaved substrate derived from
the NS4B/NS5 site (Ac-TTSTRR-pNA) had a
Km of 346 µM, kcat of 0.095 s-1 and a kcat / Km
of 275 s-1 M-1 [26] .
Recently, we have shown by a HPLC-
based assay with fluorometric detection that
the NS2B-NS3pro protease incorporating a
full-length NS2B co-factor could cleave N-
terminally dansylated 12mer peptides
mimicking native polyprotein junctions in
the absence of microsomal membranes[28] .
However, this protein was completely
inactive in autocleavage and the efficiency
of this recombinant protease with the
peptide substrate was markedly reduced
when compared to constructs incorporating
the 40-residue NS2B core sequence, likely
due to structural distortions induced by the
flanking regions of NS2B and inefficient
activation of the protease.
Currently, a detailed study on substrate
specificity of the dengue virus NS3 protease
is in progress, which uses combinatorial
libraries of internally quenched fluorogenic
peptides labelled with the aminobenzoyl /
p-nitrotyrosine reporter pair. For a substrate
peptide based on the NS3/NS4A cleavage
site, Abz-AAGRK?SLTLY(NO2)R-NH2 (?
denotes the cleavage site), a Km value of 141
µM, a kcat of 0.18 s-1 and a kcat / Km of 1262
s-1 M-1 was found, which is approximately
62
10-fold better than the best commercially
available substrate tested so far, GRR-AMC
[J. Wikberg, unpublished data]. In the near
future, these investigations will likely lead to
the identification of NS3 substrates with
optimized sequence length and improved
binding affinities, which can be applied as
sensitive probes for enzyme activity in high-
throughput inhibitor screenings.
Perspectives for inhibitor
development
Principally, every step of viral
morphogenesis, from cell-entry, uncoating,
replication and assembly of new virus
particles, is a potential target for antiviral
inhibitors. However, the molecular events in
the infectious cycle of flaviviruses such as
dengue virus are characterized only to a
very limited extent, making the design of
specific inhibitors an adventurous task. In
contrast, proteases and their inhibitors have
been intensively studied because of their
potential for the development of selective
antiviral compounds. Although tremendous
progress in the field is indicated by the
design and clinical use of antiviral drugs
against HIV (AIDS) and hepatitis C virus
proteases, the potential of dengue and
related flaviviral proteases for inhibitor
discovery is largely unexploited. In response
to the global problem of the dengue virus
epidemics, considerable efforts are now
being devoted to the development of drugs
which will eventually be suitable for a
chemotherapeutic intervention in acute
dengue diseases, not only by academic
institutions but also by pharmaceutical
companies (for example see
http://www.nitd.novartis.com).
Dengue Bulletin – Vol 28, 2004

In a first step towards a rational inhibitor
design for the dengue virus NS3 serine
protease, inhibition by synthetic peptides
mimicking uncleavable transition state
isosteres of the P6-P2’ residues of the native
polyprotein sites, was demonstrated[26] . The
peptides with an α-keto amide in place of
the scissile amide bond acted as competitive
inhibitors of the NS3 protease with Ki -
values in the micromolar range.
Replacement of the P1’-P2’ residues by a
carboxyl-terminal aldehyde in the
NS3/NS4A-derived peptide (Ac-FAAGRR-
CHO) yielded a competitive inhibitor with a
Ki of 16 µM. For the dengue virus protease,
the hexapeptides displayed Ki - values which
were only 2- to 6-fold lower than the Km -
value for the corresponding substrate, a
feature which discriminates dengue virus
NS3 from the HCV protease, where product
inhibitors had binding affinities which were
one order of magnitude lower than those for
the substrates[33,34] .
Product inhibition of the HCV NS3
protease by cleavage-site derived peptides
led to the discovery of very potent inhibitors
of this enzyme with IC50 – values in the
nanomolar range by cyclic optimization of
the inhibitor structures[33,34] . Recently, we
have shown that peptides representing non-
prime-side residues of the dengue virus NS3
protease act as competitive inhibitors of the
enzyme, whereas prime-side peptides
appeared to have negligible effects on
enzyme inhibition at concentrations >1.0
mM. (S. Chanprapaph, unpublished data). Ki
- values for hexapeptides derived from all 4
dengue virus cleavage sites were in the low
micromolar range and the best inhibitor was
based on the NS2A/NS2B site (Ac-RTSKKR-
CONH2) and gave a Ki of 12 µM. In analogy
to the HCV NS3 protease, these findings
Dengue Bulletin – Vol 28, 2004
Inhibition of NS2B/NS3 Protease
suggest the existence of a high-affinity
binding site in the non-prime region of the
enzyme and offer the prospect of
developing effective inhibitors against the
dengue virus protease by combinatorial
optimization based on the structure(s) of
native polyprotein cleavage site peptides.
However, in general, peptide-based
inhibitors exhibit poor pharmacokinetic
properties and usually the conversion of
these structures into less “peptide-like”
compounds (“peptidomimetics”) is required
to generate drug-like entities.
Inhibitor discovery for the dengue virus
NS3 protease is currently limited by the lack
of a 3-dimensional structure for the NS2B-
NS3 co-complex; however, it can be
expected that crystallographic studies and
NMR-experiments will provide more insight
into the structure and catalytic mechanism of
the enzyme in the near future. In addition,
powerful computer modelling approaches
exist which may help to obtain information
required for a rational drug design even in
the absence of crystallographic structures.
Proteochemometric modelling (PCM) is
currently explored as a tool to analyse the
structural and physicochemical properties
which are necessary for the interaction of
potential inhibitors with the dengue virus
NS3 protease target structure [35] . Preliminary
data obtained by this approach suggest that
the proteochemometric models are valid
and useful for the accelerated design of
novel inhibitors. This approach does not
only circumvent the traditional erratic dug
discovery process with its high attrition rates,
but also allows to incorporate potential
resistance of the target and the development
of ‘drug resistance – resistant’ compounds as
an initial consideration in the design process.
The existence of large conformational
63
Inhibition of NS2B/NS3 Protease
ensembles is particularly challenging in the
case of rapidly mutating viral enzymes,
where a design against a moveable target
would require large sets of corresponding
inhibitors. In the future, these problems are
likely to be addressed by ‘shotgun
approaches’ to the structure of enzyme-
inhibitor complexes and the identification of
hot spots of ‘interaction flexibility’ by the
use of fast, high-resolution methods such as
NMR. Detailed accounts on this strategy are
given in Reference 36.
Conclusions
Substantial progress has been made over the
past few years in the biochemical
characterization of the dengue virus NS2B-
NS3 two-component protease. The data,
which are available now, make the dengue
virus NS3 protease a valid molecular target
for the development of antiviral compounds.
A large repertoire of powerful methods for
inhibitor development and evaluation exists
which includes state-of-the-art technology in
organic synthesis and computer-aided
molecular design. Although there is no
suitable animal model available for dengue
virus diseases, initial screening of potential
antiviral compounds would be facilitated by
well-established insect and mammalian cell
culture systems which are useful to monitor
the effects of anti-NS3 inhibitors on the
propagation of the virus.
Moreover, alternative drug targets
which are present on the dengue virus NS3
protein can be exploited for inhibitor
development. These include the binding site
of the NS2B co-factor to the NS3 protease,
the NS3 NTPase / helicase portion and the
interaction surface of NS3 with the NS5
replicase. The presence of multiple
64
biomedical targets in the dengue virus
polyprotein would even make a therapy
feasible, which uses combinations of
different inhibitors and therefore could
minimize the risk of rapid resistance
development.
It can also be expected that progress for
inhibitors against the dengue virus protease
will be of large benefit for drug design
against related human pathogens of the
flavivirus complex such as yellow fever virus,
Japanese encephalitis virus and West Nile
virus.
However, in order to bring an effective
anti-dengue drug from the ‘bench to the
bedside’, several questions and limitations
need to be addressed. These include the
evaluation of prognostic markers for disease
severity, the pathobiochemistry of dengue
haemorrhagic fever and shock syndrome,
the problem of selectivity against
pharmacologically relevant human proteases
such as furin and the risk of adverse effects.
Potential complications may also arise from
the presence of four related dengue
serotypes in the case that their NS3
proteases show marked differences in their
inhibition profiles. Intensive efforts and
sustained multidisciplinary research is
required in the future to cope with the
challenging task of a causative treatment for
dengue virus diseases.
Acknowledgements
We thank Dr J. Wikberg, Department of
Pharmaceutical Biosciences, Uppsala
University, Sweden, for intensive discussions
and access to data prior to publication. This
work was supported by grants from the
Thailand Research Fund (TRF).
Dengue Bulletin – Vol 28, 2004

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