AIDS2012 TUAA0301

Pre-clinical Evaluation of HIV Replication Inhibitors that Target the HIV Integrase-

LEDGF/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 TherapyKU Leuven

Belgium

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)

LEDGF/p75 is a novel target for antiviral therapy

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 antiviralsName Structure LEDGF/p75-

integrase

MTT/MT-4

IC50 [µM] EC50 [µM] CC50 [µM]SI

CX051681.37±0.36 2.35±0.28 59.8±0.5 25

CX050450.58±0.30 0.76±0.08 72.2±5.15 95

CX144420.046±0.012 0.069±0.003 96.0±16.0 1391

NO

OHCl

S NO

OH

S NO

OHO

1tan28a566028 52tan28a566028 0tan28a566028 0tan19a5660190tan8a56608

0tan28a566028

0tan19a566019

0tan9a56609

0tan29a566029

0tan19a566019

0tan10a566010

0tan30a566030 Ral-tegravirSeries 1Series 2

log [compound]

% in

hibi

tion

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?

compound EC50 [nM]

CX05168 1136,5±625

CX05045 3093±1784

CX14442 123±0,6

-3 -2 -1 0 1 20

20000

40000

60000

80000

CX05168CX05045CIM055138

log [µM]

rela

tive

coun

ts

LEDGINs stabilize the dimer interface of integrase and increase the melting temperature of the integrase multimer.

Oligomerization of HIV-integrase

compound TM (°C)

DMSO 48,1

CX05168 54,5

CX05045 57,3

CX14442 62,5

Melting of HIV-integrase

(Christ, et al., AAC, 2012)

LEDGINs inhibit HIV replication at the integration step (TOA)

Raltegravir, elvitegravir and LEDGINs profile nearly identical in Time of Addition studies.

0tan28a566028 0tan9a56609 0tan19a566019 0tan29a5660290tan28a566028

0tan19a566019

0tan9a56609

0tan29a566029

0tan19a566019

0tan10a566010

0tan30a566030tenofovir elvitegravirCX14442 zidovudine

Hours post infection

Per

cent

inhi

bitio

n

(Christ, et al., AAC, 2012)

LEDGINs activity against resistance mutants

LEDGINs are active against a wide range of INSTI resistant mutants. LEDGINs are active against a broad range of HIV-1 subtypes.

CX05045raltegravir

LEDGIN raltegravir capsid inhibitor

(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

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.

Production of IIIB Infectivity of IIIB

DMSO raltegravir ritonavir CX05045

(Christ, et al., AAC, 2012)

DMSO raltegravir ritonavir CX05045

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 LeuvenMolecular 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 Smith-Burchnell, 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.