Evidence for Host Drug Targets Essential for Dengue Virus Capsid Formation
Marissa Baker-Wagner1, Nicole Wilcott2, Yoko Marwidi1, Shao Feng Yu1, Debendranath Dey1, Bruce Onisko1, Katie Barlow1, Shalini Potluri1, Christine Sahlman1, Alfredo Calayag1, Vishwanath R. Lingappa1, Pamela Glass3, Michael Farmer1, Clarence R. Hurt1, William Hansen1
1Prosetta Bioconformatics, Inc, San Francisco, CA, United States/ 2CUBRC, INC, Buffalo, NY, United States/ 3Virology Division, USAMRIID, Ft Detrick, MD, United States
EC99
EC50
EC99
EC50
EC99
140
140
<0.05uM
<0.05uM
<0.05uM
<1uM
1uM
1uM
10uM
120
120
>20uM
>20uM
ND
ND
>20uM
>20uM
PAV-285
<1uM
7.5uM
1uM
>20uM
ND
ND
<25uM
25uM
PAV-772
<20uM
20uM
<10uM
>10uM
<2uM
10uM
10uM
>10uM
PAV-827
<10uM
<10uM
10uM
>10uM
ND
ND
ND
ND
1
0.5
100
80
60
100
80
60
40
40
20
20
0
0
PAV-899
PAV-790
PAV-285
PAV-772
PAV-827
0uM
10uM
25uM
50uM
10uM
0uM
2.5uM
10uM
25uM
<7.5uM
0uM
2.5uM
10uM
25uM
PAV-790
RFUs (%0uM)
EC50
0uM
5uM
10uM
20uM
1.5
160
EC99
Legend to Figure 4. Compounds representing several pharmacophores identified through the Prosetta plate screen
were compared for their efficacy against live DENV in cell culture and against other members of family Flaviviridae
(WNV and HCV). In addition, as we have observed a strong correlation between activity against the Flaviviridae
and the Togaviridae, their efficacy against VEEV, a member of the latter family was assessed as well. Some
remarkable observations are that 1) PAV-899 and PAV-285 shows strong efficacy against all viruses tested,
illustrating the propensity of some of our compounds to be pan family and more in its anti-viral spectrum; 2) PAV790 is active only on DENV and not on WNV or VEEV, illustrating that a degree of viral specificity can be observed
within this pharmacophore as well; 3) PAV-772 appears potent against all of the Flaviviridae, but without strong
effect on VEEV; 4) PAV-827, the structure being disclosed, may be similar to PAV-790 in its relative DENV
specificity.
2
160
0uM
2.5uM
10uM
25uM
2.5
PAV-899
PAV-790
PAV-285
PAV-772
PAV-827
Legend to Figure 5. Some representative hits from the DENV screen on the HCV screen. As can be seen,
these two members of family Flaviviridae give distinctive differences – but share in common that they all
would have been scored as hits in either screen.
0uM
5uM
10uM
20uM
0uM
5uM
10uM
20uM
0uM
5uM
10uM
20uM
PAV-191
PAV-772
PAV-668
PAV-092
Cmpnd
D
PAV-285
0uM
5uM
10uM
20uM
0uM
5uM
10uM
20uM
0
0uM
5uM
10uM
20uM
Legend to Figure 1. Cell-free translation of
DENV core transcript reveals the expected
12kDa protein in a discrete complex in fraction (f)
5 on sucrose step density gradients (ssg).
3
VEEV
0uM
5uM
10uM
20uM
Percent Control RFU/100
3.5
HCV
0uM
5uM
10uM
20uM
PAV-899 <0.05uM
4
Legend to Figure 2. Translation products of DENV core
polypeptide were analysed by sucrose step gradient velocity
sedimentation (ssg), polyacrylamide gel electrophoresis in
sodium dodecyl sulfate (SDS PAGE), with autoradiography and
quantitative densitometry at various times after start of protein
synthesis. The parameters expected to affect an enzymatic
process (time, temperature, substrate and host factor
concentration) were varied and the consequences for flux of
putative intermediates assessed.
EC50
WNV
HCV NP
0uM
5uM
10uM
20uM
PAV #
4.5
( Fraction )
DENV NP
DENV
5
DENV vs. HCV Cell-Free Screen
Efficacy of Prosetta Drugs on Live DENV in Cell Culture
0uM
2.5uM
10uM
25uM
A Diversity of Phenotypes of DENV core Fluorescence
Readout in the Capsid Assembly Plate Screen
0uM
5uM
10uM
20uM
Time Course Reveals Putative DENV
Capsid Assembly Intermediates
% of total material
By analogy to previous studies for Hepatitis B virus, HIV, and Hepatitis C virus,
we established a cell-free system involving de novo protein biogenesis that
appears to faithfully carry out critical steps in the assembly of Dengue virus
capsids. The protein synthesis-linked capsid assembly system was converted
into an ELISA-based screening platform for identification of small molecules
that interfere with proper Dengue virus capsid formation. This screen
potentially can identify molecules acting either directly or indirectly, via
interference with essential host factors, anywhere in the assembly pathway. A
number of small molecules conforming to Lipinski’s Rules were identified as
hits likely acting at diverse steps in the capsid assembly pathway and by
different mechanisms. This hypothesis is based on evidence to be presented
that the activity of some of these molecules results in aberrant capsids by
several different criteria including resistance to digestion by proteases and
changes in buoyant density, compared to non drug-treated controls. When
tested against live Dengue virus in cell culture, a number of these compounds
were found to be robustly active, resulting in multilog drop in plaque forming
unit (pfu) titer in the nanomolar to low micromolar range. These active
molecules were sorted by chemical class, activity, and toxicity. A total of 11
chemical classes (pharmacophores) were found to be potent (EC 50<7.5uM)
and non-toxic (TI>10). These findings provide strong support for the hypothesis
that critical steps in Dengue virus capsid formation are faithfully re-created in
the cell-free system. The targets of those drugs not acting directly on the
capsid protein are promising candidates for essential host factors in the
Dengue virus life cycle.
Cell-free Translation of DENV
core Reveals an Apparent
Assembled Complex by ssg
RFUs (%0uM)
Abstract
PAV-790
Enhanced Protease Sensitivity Reveals Aberrant Capsid
formation as One Mechanism of Prosetta Drug Action
Bridging the Gap: Next Generation Cell-free Systems and
Future Traction on Protein-Protein Interactions
30
25
Potential Aberrant DENV Capsid Inducing Compound
CFPS of DENV core +DMSO
Fractionation and Reconstitution of Host
Proteins Essential for DENV Capsid Formation
CFPS of DENVcore + PAV-827
20
Proteins Bound to PAV-772
Successfully Used for
Reconstitution of DENV
Capsid Assembly
15
DENV
10
5
0
A. Authentic irradiation
inactivated DENV Capsids
25.0
C. Subcellular fraction
complementation
20.0
PAV-790
15.0
PAV-285
10.0
PAV-092
5.0
PAV-827
200 kD
0.0
Reconstitution of DENV capsid assembly
by combining dialyzed drug column
eluate with flow through
DENV capsid assembly in starting extract
1 2 3 4 5 6 7 8 9 10 11
40
116 kD
98 kD
B. Cell-free DENV capsids made
by manipulation of whole extract
66kD
45 kD
35
M 1
2 3 4 5 6 7 8 9 10 11 T
1 M 2 3 4
5 6 7 8 9 10 11 T
30
25
29 kD
20
15
+ PAV-790
+ PAV-092
Legend to Figure 6. As can be seen here, some compounds, such as PAV-790 and PAV-092 appear to
cause a block in DENV capsid formation such that the putative early assembly intermediate seen in
fraction 2/3 accumulates upon drug treatment in the cell-free system.
Legend to Figure 10. To test the hypothesis that the action of numerous Prosetta drugs is to
generate an aberrant DENV capsid, as has been observed for other viruses, cell-free
assembled capsids (sucrose gradient fractions) were prepared in the presence of drug or
DMSO control and subjected to protease digestion to assess their integrity. As can be seen,
capsids made in the presence of all three of the drugs tested were found to be aberrant by
virtue of their extreme protease sensitivity compared to DMSO control capsids.
10
Blockade of DENV Capsid Assembly by Small Molecules
in the Prosetta Cell-Free System
+ DMSO
E. Complementation with a
specific host factor
suspected to be involved in
a rate-limiting step in capsid
maturation
5
0
1
Legend to Figure 7. As can be seen here, some compounds,
such as PAV-827, whose structure is disclosed here, not only
do not block assembly, they seem to promote assembly – but
possibly off the pathway of authentic capsid formation. Thus
the amount of material in fractions 5/6 in the drug-treated
sample is the same or greater than was observed in the
presence of DMSO control. Given that the compound has
been shown to have potent antiviral activity, how can we
account for this lack of a simple mechanistic explanation? One
possibility is that the action of many of these compounds is to
generate an aberrant capsid (which renders the virus noninfectious).
Lack of DENV capsid assembly in drug
column flow thru
Minimal DENV capsid assembly in
drug column flow thru reconstituted
with control column eluate
2
3
4
5
6
7
8
9 10 11
Reconstitution as above with dialyzed
drug column eluate from rat liver
Legend to Figure 8. Extract competent for
cell-free translation was passed over PAV-772
drug column, washed extensively and the
dialyzed free drug eluate added back to the
flow through or not and then used for cell-free
translation.
Newly synthesized DENV is
unable to assemble in the absence of eluate
or with control eluate but does so when
reconstituted with dialyzed eluate from either
the starting extract or rat liver. The ability to
fractionate and reconstitute using an extract
that has not yet engaged in synthesis of the
viral capsid protein, is a powerful functional
tool for biochemical dissection of the
pathways of catalyzed capsid assembly.
D. Manipulation of energy
substrates
Legend to Figure 11. Capsid assembly represents a series of steps that are themselves nested within an even more complex series
of steps of the larger viral lifecycle. Our cell-free system reconstitutes a number of these steps in a manner that makes them
amenable to biochemistry and drug discovery that is not easily achieved by other means. This does not mean that the events
reconstituted in our system are the complete pathway – indeed, even a partial reconstitution of a novel pathway could account for
the efficiency of anti-viral drug discovery we have observed. Authentic (irradiated) DENV capsids migrate in a different position in
sucrose gradients than the complexes initially formed (fraction 5/6), suggesting that additional steps may be possible to achieve in
reconstitution of the pathway of DENV capsid assembly. In the course of our studies we have advanced to what we believe is the
5th generation iteration of the cell-free system. Those subsequent improvements include variants that dissociate synthesis from
assembly, that complement the steps readily achieved with more difficult steps not easily detected at steady state or not easily
reconstituted without manipulation of either enzymes or substrates involved in the reaction. Thus, manipulation of whole extract (B),
subcellular fraction (C), energy substrates (D), and specific proteins (E) are able to generate structures that appear more faithful to
the authentic DENV capsid (A).
Summary and Future Plans
Legend to Figure 9. Coomassie staining
of the eluate reveals a set of unique
protein bands not observed with the
control column eluate (same resin and
same method of blocking, but lacking the
ligand PAV-772). Mass spectrometry of
these proteins together with chemical
crosslinking of the nearest neighbor will
allow identification of the minimal drug
target and the composition of the
machine likely involved in catalyzed
capsid formation.
• Prosetta takes a biochemical and cell biological approach to a classical issue in virology, namely the formation of the capsid.
• Each viral family displays distinctive characteristics including shared and distinctive features in the Prosetta platform.
• The fruits of this approach have been a plethora of novel drugs
• The promise for the future is insight into the targets and mechanism that are not easily achieved by other experimental means.
• Small molecules have been identified through the Prosetta platform that represent promising early stage molecules for an anti-DENV therapeutic development program.
• These molecules most likely target host protein since fractionation of extracts on drug affinity columns and reconstitution with dialyzed free drug eluates has been achieved. This occurs prior to viral
capsid protein expression.
• At least some of these molecules are likely to exert their anti-viral effect through generation of aberrant capsids that are non-infectious due to misfiring of the machinery of catalytic capsid assembly.