carbohydrate-based strategies of antiviral therapeuticscv-n 0.003 0.002 0.017 prm-a 3.4 1.8 5.0 uda...
TRANSCRIPT
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Carbohydrate-based strategies of antiviral therapeutics
Prof. Jan Balzarini
Rega Institute for Medical ResearchB-3000 Leuven, Belgium
VII Jornadas de la Sociedad Espanola deQuímica Terapéutica, Sitges, 19-21 October 2006
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Combination therapy for HIV infections
NRTIsZidovudineDidanosineZalcitabineStavudineLamivudineAbacavirEmtricitabine
NNRTIsNevirapineDelavirdineEfavirenz
PIsSaquinavirRitonavirIndinavirNelfinavirAmprenavirLopinavirAtazanavirDarunavir
NtRTIsTenofovir
FIsEnfuvirtide
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Binding to CD4
Binding to coreceptor
Membrane fusion
1 2 3
HIV entry is triggered by receptor engagement
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HIV entry into target cells
• Multi-step process
• Timely and locally ordered exposure of previously hidden entry domains within gp120/gp41
• Hidden domains protected from immune attack ! (These epitopes are only exposed when the virus is close enough to the cell membrane to initiate entry.)
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HIV entry into target cells
• Interaction gp120 with specific receptor CD4 ⇒ limited host range of virus
• ∆ conformation gp120⇒ hidden conserved epitope binds to (chemokine) co-receptor– CXCR4 (SI or X4)– CCR5 (NSI or R5)– others
• ∆ conformation of gp120⇒ exposure of gp41⇒ pre-fusogenic → fusogenic conformation
(pre-hairpin) (hairpin)• Fusion between viral and cellular membranes
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Entry inhibitors of HIV
• Attachment (adsorption) inhibitors– Block initial binding of virus to the cells
• Co-receptor binding inhibitors– Interfere with co-receptors
• Env gp120 binding inhibitors– Interfere with gp120
• Fusion inhibitors– Prevent the fusion process between viral and cell
membrane
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Why HIV entry inhibitors ?
• New target, different from RT and protease• Other toxicity profile• Other drug resistance profile
– Select for characteristic HIV mutations in “entry target”
– Suppress HIV with other mutated targets• Systemic use/microbicidal use
– Block cell-free virus infection/cell to cell infection
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Entry inhibitors
Focus on carbohydrate-binding agents (CBA)
Rational:• HIV gp120 envelope is highly glycosylated (~ 50%)• Envelope glycans required !
– Proper folding gp120 envelope– Efficient entry into target cells– Efficient DC-SIGN-directed transmission of HIV to T-
lymphocytes– Hiding immunogenic epitopes at gp120 (escape immune
system)
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Speculations, wishful thinking, or reality ?
• CBA may afford an efficient blockade of the entry process
• CBA may prevent efficient transmission from dendritic cells (DC-SIGN) to T-lymphocytes
• CBA may force HIV to delete gp120 glycans to escape drug pressure
• CBA may, indirectly, boost the immune system to produce specific Nabs against mutant virus strains, resulting in “self-vaccination”
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To prove the CBA concept: initial focuss on plant lectins
(Collaboration with E. Van Damme & W. Peumans, UGent, Belgium & D. Schols, Rega Institute, Leuven, Belgium)
HHA GNA LOA UDA
(Man) (Man) (Man) (GlcNAc)
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GNA tetramer bound to 12 MeMan molecules
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Pradimicin A
O
OHHO
HO
H3CO
OH O
O OHHO
OH
CH3
O
CO NH CH COOH
CH3
O CH3HO
NHCH 3O
... but also the small-size antibiotic Pradimicin A, known to recognize α(1,2)mannose oligomers ...
(Collaboration with T. Oki & Y. Igarashi, Japan)
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C
35027019011030
0
170
380
590
800
RU
A
170
380
590
800
0
B
160
560
960
1360
Time (s)
D
160
560
960
30 110 190 270 350 22000 0
PRM
A
H
HA
M. Rusnati & A. Bugatti, Brescia, Italy
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Effect of CBA on ...HIV infection of T-lymphocytes and
macrophages
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Antiretroviral activity of CBA in cell culture
0.0170.0020.003CV-N
5.01.83.4PRM-A0.1620.3300.140UDA
0.0750.0010.004EHA0.0300.0030.004LOA0.0340.0180.031CA0.0200.0130.009NPA0.0100.0160.006HHA0.0420.0110.018GNA
SIVmac(MT-4)
HIV-2(ROD)(CEM)
HIV-1(IIIB)(CEM)
EC50a (µM)CBA
aEffective concentration, or compound concentration required to inhibit virus-induced cytopathicity in cellculture.
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Inhibitory activity of CBA against HIV strains and clinical HIV-1 clade isolates in PBMC cultures
N.D.2.63.40.83N.D.2.6134.13.7102.72.6PRM-A
1.01.00.50.561.01.00.610.621.01.00.560.97UDA
0.0130.160.0020.0130.0130.0770.100.100.030.160.040.13CV-N
0.0880.190.0290.0880.260.740.500.090.420.960.230.44CA
0.180.120.0220.0280.0240.820.090.240.980.880.110.58HHA
0.190.130.0660.0280.038≥ 20.500.38> 0.42.00.340.54GNA
HIV-2BV-5061W
(X4)
BaL
(R5)
IIIB
(X4)
NL4.3
(X4)
OBCF06
(X4)
GBCF-DIOUM
(R5)
FBZ163(R5)
EID12(R5)
DUG270
(X4)
CETH2220
(R5)
BUS2(R5)
AUG273
(R5)
CBA
EC50a (µM)
a50% Effective concentration required to inhibit HIV replication in cell culture.
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Effect of mannan on the anti-HIV-1 activity of CBA's in CEM cell culture CBA EC50
(µg/ml)
as such + mannan 2.5 mg/ml GNA 0.45 ± 0.30 27 ± 11 HHA 0.50 ± 0.44 26 ± 8.7 CV-N 0.019 ±0.018 0.35 ± 0.21 PRM-A 6.0 ± 2.8 32.5 ± 10
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Effect of CBA on ...syncytia formation between (persistently)HIV-1-infected cells and T-lymphocytes
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A cocultivation assay between persistently HIV-1-infected HUT 78 cells and SupT1 cells
Compound EC50a (µM)
GNA 0.12 ± 0.06 HHA 0.09 ± 0.05 CV-N 0.02 ± 0.0 UDA 0.51 ± 0.26 Pradimicin A 4.1 ± 1.6 a 50% effective concentration or compound concentration required to inhibit syncytium formation between HUT-78/HIV-1 and Sup T1 cells by 50 %
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Effect of CBA on ...DC-SIGN-directed capture of HIV-1
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0
20
40
60
80
100
5 1 0.2 5 1 0.2 0.04 10 2 0.4 0.08GNA (µM) HHA (µM) CA (µM)
0
20
40
60
80
100
30 6 1 60 12 250 50 250 50
Compound concentration
UDA (µM) PRM-A (µM)
HIV
-1 c
aptu
re b
y R
aji/D
C-S
IGN
cel
ls (p
erce
nt o
f inh
ibiti
on)
DS-5000 (µg/ml) PVAS (µg/ml)
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Effect of CBA on ...DC-SIGN-directed transmission of HIV-1 to
T-lymphocytes
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without HIV-1 plus HIV-1
Raj
i/DC
-SIG
N +
C81
66
C
8166
Raj
i/DC
-SIG
N
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Raji/DC-SIGN +NPA + HIV-1
+ C8166 day 2 p.i.wash
1 µM
0.04 µM
0.0016 µM
0.2 µM
0.008 µM
0 µM
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Inhibition of DC-SIGN-directed capture of HIV-1 and transmission to T-lymphocytes by CBA
activeactivePRM-A
3.31.1UDA
0.0040.003CV-N
0.060.10CA
0.840.36GNA
0.640.17HHA
EC50 (µM)IC50 (µM)
Raji/DC-SIGN + HIV-1 + C8166 co-culture
Raji/DC-SIGN + HIV-1Compound
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Effect of CBA on ...Selection of mutant HIV-1 strains in cell
culture
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Resistance selection of HIV-1 IIIB against UDA, HHA, GNA and Nevirapine
0
50
100
150
200
250
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111
Subcultivation number
UDA,
HHA
and
GNA
dru
g co
ncen
trat
ion
(µg/
ml)
0
2,5
5
7,5
10
12,5
Nev
irap
ine
drug
con
cent
ratio
n (µ
g/m
l)
HIV-1/UDA-1
HIV-1/UDA-2
HIV-1/HHA
HIV-1/GNA
HIV-1/Nevirapine
Drug pressure against HIV-1(IIIB) by nevirapine, HHA, GNA or UDA
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Man Man
Man
Man
Man
Man
Man
Man
Man
GlcNAc
GlcNAc
Asn
α-1,2α-1,2 α-1,2
α-1,2
α-1,3
α-1,3 α-1,6
α-1,6
β-1,4
β-1,4
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Man Man
Gal
GlcNAc
Gal
GlcNAc
Gal
Man
Fuc-GlcNAc
GlcNAc
Asn
GlcNAc
SA SA SA
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N-Glycosylation site mutations in gp120 of mannose-binding plantlectin-exposed HIV-1 strains
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PRM-A-selected N-glycan deletions in HIV-1 gp120
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Summary CBA-selected N-glycan deletions in HIV-1 gp120
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Conclusions CBA pressure against HIV-1 in cell culture
• Predominant selection of glycan deletions in gp120, but not in gp41
• Preference for high-mannose type glycan deletions
• Multiple glycan deletions are required for significant phenotypic resistance
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Conclusion
• CBA are potent inhibitors of HIV but also HCV !• CBA are not inhibitory to many other enveloped
viruses
CBA show pronounced selectivity in their antiviralaction
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Potential pittfalls of CBA as therapeutics
• Mitogenic ?• RBC agglutination ?• Stimulation of differentiation markers ?• Immunological response ?• Glycans of cellular proteins targeted as well ?• Specificity (therapeutic window) ?
Challenge: select/design CBA that are preferentially targettingglycoproteins of the pathogen !
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HIV infection of T-lymphocytes& macrophages
Selection ofHIV strains with glycan deletions
in gp120
CBA
Syncytia formationbetween persistently
HIV-infected cellsand T-lymphocytes
HIV transmissionto T-lymphocytes
through HIV captureby DC-SIGN
HIV neutralization bytriggering neutralizingantibody production to
uncovered gp120 epitopes
?
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Conclusions CBA concept - 1
CBA concept is entirely new:• Unique target interaction: carbohydrates (glycans)• No need for cellular uptake nor metabolic conversion• Unique resistance profile: glycan deletions• No cross-resistance to other drugs• Ratio number of drug molecules attached to target
glycoprotein is >> 1• High genetic barrier: multiple mutations required before
resistance development
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• Accumulation of glycan deletions may compromise the correct folding and conformation of the glycoprotein, resulting in attenuated infectivity (fitness) and transmission
• Mutations expected to trigger the immune system against unhidden, exposed, immunogenic epitopes
• First approach that may combine drug therapy and induction of specific Nab response (“self-vaccination”)
Conclusions CBA concept - 2
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• To be applied to chronic virus infections with glycosylated envelope (i.e. HIV, HBV, HCV, ...)
• To be applied to acute virus infections with glycosylated envelope (i.e. influenza virus, corona (SARS) viruses)
Conclusions CBA concept - 3
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Acknowledgments
Rega Institute for Medical Research,Leuven, Belgium
Kurt VermeireKatrien FrançoisJoeri AuwerxKristel Van LaethemDominique ScholsJan Balzarini
University of Brescia, ItalyM. RusnatiA. Bugatti
University of Ghent, BelgiumW. PeumansE. Van Damme
Toyama Prefectural University, Tokyo, Japan
Y. IgarashiT. Oki