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

Combination therapy for HIV infections

NRTIsZidovudineDidanosineZalcitabineStavudineLamivudineAbacavirEmtricitabine

NNRTIsNevirapineDelavirdineEfavirenz

PIsSaquinavirRitonavirIndinavirNelfinavirAmprenavirLopinavirAtazanavirDarunavir

NtRTIsTenofovir

FIsEnfuvirtide

Binding to CD4

Binding to coreceptor

Membrane fusion

1 2 3

HIV entry is triggered by receptor engagement

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.)

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

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

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

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)

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”

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)

GNA tetramer bound to 12 MeMan molecules

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)

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

Effect of CBA on ...HIV infection of T-lymphocytes and

macrophages

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.

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.

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

Effect of CBA on ...syncytia formation between (persistently)HIV-1-infected cells and T-lymphocytes

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 %

Effect of CBA on ...DC-SIGN-directed capture of HIV-1

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)

Effect of CBA on ...DC-SIGN-directed transmission of HIV-1 to

T-lymphocytes

without HIV-1 plus HIV-1

Raj

i/DC

-SIG

N +

C81

66

C

8166

Raj

i/DC

-SIG

N

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

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

Effect of CBA on ...Selection of mutant HIV-1 strains in cell

culture

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

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

Man Man

Gal

GlcNAc

Gal

GlcNAc

Gal

Man

Fuc-GlcNAc

GlcNAc

Asn

GlcNAc

SA SA SA

N-Glycosylation site mutations in gp120 of mannose-binding plantlectin-exposed HIV-1 strains

PRM-A-selected N-glycan deletions in HIV-1 gp120

Summary CBA-selected N-glycan deletions in HIV-1 gp120

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

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

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 !

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

?

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

• 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

• 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

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