Transcript AIDS2012 TUAA0301 Pre-clinical Evaluation of HIV Replication Inhibitors that Target the HIV IntegraseLEDGF/p75 Interaction Frauke Christ, Chris Pickford, Jonas Demeulemeester, Stephen Shaw, Belete Desiemmie, Caroline.

AIDS2012
TUAA0301
Pre-clinical Evaluation of HIV Replication
Inhibitors that Target the HIV IntegraseLEDGF/p75 Interaction
Frauke Christ, Chris Pickford, Jonas Demeulemeester, Stephen Shaw, Belete
Desiemmie, Caroline Smith-Burchnell, Scott Butler, Mike Westby, Zeger Debyser
Molecular Virology and Gene Therapy
KU Leuven
Belgium
LEDGF/p75 is a novel target for antiviral therapy
LEDGF/p75 is a co-factor of HIV integrase
(Cherepanov et al, JBC, 2003) that tethers the
provirus to the cellular genome.
The interface of LEDGF/p75 and integrase is well
defined (Cherepanov et al. PNAS, 2005)
Overexpression of the LEDGF/p75 integrase
binding domain (IBD) inhibits HIV replication (De
Rijck et al., JVI, 2006)
LEDGINs, first in class allosteric integration inhibitors
bind to the LEDGF/p75 binding site on integrase. (Christ,
et al., Nat. Chem. Biol. 2010)
LEDGINs, first-in-class antivirals
1.84 Å co-crystal structure
LEDGINs are a novel class of HIV-replication inhibitors
designed on the basis of a pharmacophore model.
By combination of medicinal chemistry and structural
biology different series of compounds have been
developed with activities in the low nanomolar range
and selectivity >5000.
LEDGINs block the LEDGF/p75-IN interaction and bind to an
allosteric site on integrase.
(Christ, et al., Nat. Chem. Biol. 2010)
LEDGINs, first-in-class antivirals
Name
Structure
LEDGF/p75-
MTT/MT-4
120
integrase
IC50 [µM]
EC50 [µM]
CC50 [µM]
SI
1.37±0.36
2.35±0.28
59.8±0.5
25
CX05168
% inhibition
100
Raltegravir
Series 1
Series 2
CX14442
CX5045
80
60
40
20
CX05045
0.58±0.30
0.76±0.08
72.2±5.15
95
0
-20
0.0001
CX14442
0.01
1
log [compound]
0.046±0.012
0.069±0.003
96.0±16.0
1391
 More potent LEDGINs have been synthesized allowing for a detailed study of
the mechanism of action.
 All compounds binding to the LEDGF/p75 binding pocket inhibit LEDGF/p75
binding and strand transfer activity of integrase.
(Christ, et al., AAC, 2012)
What is the underlying allosteric mechanism?
 LEDGINs potently inhibit the strand transfer reaction.
 Inhibition of the strand transfer reaction is more efficient if compounds are added
to integrase before the DNA substrate.
(Christ, et al., AAC, 2012)
What is the underlying allosteric mechanism?
Oligomerization of HIV-integrase
Melting of HIV-integrase
relative counts
80000
60000
40000
CX05168
CX05045
CIM055138
20000
0
-3
-2
-1
0
1
2
log [µM]
compound
TM (°C)
compound
EC50 [nM]
DMSO
48,1
CX05168
1136,5±625
CX05168
54,5
CX05045
3093±1784
CX05045
57,3
CX14442
123±0,6
CX14442
62,5
 LEDGINs stabilize the dimer interface of integrase and increase the melting temperature
of the integrase multimer.
(Christ, et al., AAC, 2012)
LEDGINs inhibit HIV replication at the integration step (TOA)
120
tenofovir
CX14442
raltegravir
Percent inhibition
100
elvitegravir
zidovudine
ritonavir
80
60
40
20
0
0
5
10
15
20
Hours post infection
25
30
 Raltegravir, elvitegravir and LEDGINs profile nearly identical in Time of Addition studies.
(Christ, et al., AAC, 2012)
LEDGINs activity against resistance mutants
CX05045
raltegravir
LEDGIN raltegravir capsid inhibitor
 LEDGINs are active against a wide range of INSTI resistant mutants.
 LEDGINs are active against a broad range of HIV-1 subtypes.
(Christ, et al., AAC, 2012)
LEDGINs activity against resistance mutants
 LEDGIN resistance mutants were
identified in serial passaging experiments
with HIV-1 (NL4-3)
 Mutations were introduced to the IN gene
of NL4-3 by site directed mutagenesis
 Series 2 and Series 3 are significantly
less susceptible to resistance mutations
of series 1
 None of the LEDGINs lose activity in the
presence of STI resistance mutations
Combination of LEDGINs and INSTIs
LEDGINs and raltegravir act additive with a tendency towards synergy when combined
for therapy.
(Christ, et al., AAC, 2012)
LEDGINs impair the infectivity of viral particles
Production of IIIB
DMSO raltegravir ritonavir CX05045
Infectivity of IIIB
DMSO raltegravir ritonavir CX05045
 Alike for raltegravir mature viral particles are produced in the presence of LEDGINs
 but the viral particles produced in presence of CX05045 are impaired in their
infectivity
 LEDGINs do not only inhibit the provirus formation but alo the infectivity of newly
produced viral particles.
(Christ, et al., AAC, 2012)
Summary
 LEDGINs are a novel promising class of inhibitors potently blocking HIV replication.
LEDGINs have multimodal mechanism
direct inhibition of the LEDGF/p75-IN interaction
allosteric inhibition of the catalytic activity of integrase
Viral particles produced in the presence of LEDGINs are severly
impaired for their infectivity.
 LEDGINs act at the same step of HIV replication like INSTIs and potently block INSTI
resistant strains.
Due to a lack of cross-resistance and additive effects of LEDGINs and raltegravir in
combination experiments LEDGINs hold great promise for further clinical development.
Thank you….
KU Leuven
Molecular Medicine: Belete A. Desimmie, Jonas Demeulemeester,
Barbara Van Remoortel, Nam Joo Van der Veken, Zeger Debyser
CD3 Leuven, CISTIM Leuven vzw: Damian Marchand, Arnaud Marchand,
Dorothée Bardoit, Wim Smets, Patrick Chaltin
Pfizer WRD
Sandwich Research: Chris Pickford, Stephen Shaw, Caroline SmithBurchnell, Jenny Middleton, Kevin Whitby, Scott Butler, Mike Westby
Chemistry: David Pryde, Florian Wakenhut, Karl Gibson
We thank ViiV Healthcare, the European
Commission and the IWT for their funding and
support.