1. 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 Therapy KU Leuven Belgium

2. 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

3. 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)

4. LEDGINs, first-in-class antivirals NameStructure LEDGF/p75- integrase MTT/MT-4 IC 50 [µM] EC 50 [µM] CC 50 [µM] SI CX05168 1.37±0.362.35±0.2859.8±0.525 CX05045 0.58±0.300.76±0.0872.2±5.1595 CX14442 0.046±0.0120.069±0.00396.0±16.01391  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)

5. 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)

6. What is the underlying allosteric mechanism? compoundEC 50 [nM] CX051681136,5±625 CX050453093±1784 CX14442123±0,6  LEDGINs stabilize the dimer interface of integrase and increase the melting temperature of the integrase multimer. Oligomerization of HIV-integrase compoundTM (°C) DMSO48,1 CX0516854,5 CX0504557,3 CX1444262,5 Melting of HIV-integrase (Christ, et al., AAC, 2012)

7. LEDGINs inhibit HIV replication at the integration step (TOA)  Raltegravir, elvitegravir and LEDGINs profile nearly identical in Time of Addition studies. Hours post infection Percent inhibition (Christ, et al., AAC, 2012)

8. 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. CX05045 raltegravir LEDGIN raltegravir capsid inhibitor (Christ, et al., AAC, 2012)

9. 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

10. Combination of LEDGINs and INSTIs  LEDGINs and raltegravir act additive with a tendency towards synergy when combined for therapy. (Christ, et al., AAC, 2012)

11. 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 IIIBInfectivity of IIIB DMSO raltegravir ritonavir CX05045 (Christ, et al., AAC, 2012) DMSO raltegravir ritonavir CX05045

12. 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.

13. 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 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.