HIV- -1 Integrase: 1 Integrase: HIV not just an not just an other HIV enzyme other HIV enzyme Vandekerckhove Linos MD, PhD Dpt. of Infectious Diseases University Hospital Ghent Belgium
IN a 32kD enzyme IN a 32kD enzyme 288 1 N-terminal 50 CCD: DDE 112 C-terminal 288
Vandekerckhove et al., Reviews in Antiviral Therapy 2008
T BLOCK after NI T T (before) NI T BLOCK T T Z T T T Integrated DNA Viral DNA 2-LTR circles
Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen Brass et al., Science. 2008 Feb
HIV- - cofactors cofactors HIV Binding partner (direct or indirect) Data on mechanism of action Effect of interruption of the binding between the cofactor and integrase (knock-dowm) Data on the structure of the interaction surface (Protein protein interaction) Antiviral effect (of a small compound) Selection for resistance upon interfering New ART with the interaction (J. Coffin rule) target
LEDGF/p75, a novel Binding partner of HIV Binding partner of HIV- -IN IN LEDGF/p75, a novel 1 91 148 156 325 429 530 aa 347 PWWP AT NLS IBD p52 p75 Identified as binding partner of HIV- -1 IN 1 IN Identified as binding partner of HIV (Cherepanov Cherepanov et al., 2003; et al., 2003; Maertens Maertens et al., 2003) et al., 2003) ( Lentiviral specific and increasing affinity of IN for specific and increasing affinity of IN for Lentiviral DNA DNA (Busschots Busschots et al., 2005) et al., 2005) (
Q-PCR to pinpoint Reverse transcription the replication block 293T 50 293TeGFP DNA copies / cell 40 293T-eGFPIBD 30 293T-eGFP Δ 325 Non-Integrated Nuclear Viral DNA 20 293T-eGFP Δ 325D366A 293T 1,4 10 2LTR Circles / Cell 1,2 293TeGFP 1 0 0,8 293T-eGFPIBD 8 24 48 72 0,6 293T-eGFP Δ 325 Hours Post Infection 0,4 0,2 293T- 0 eGFP Δ 325D366A 0 8 24 48 72 Hours Post Infection Integrated proviral DNA Integrated copies/cell 2 1,5 1 0,5 0 eGFP Δ 325D36 eGFP Δ 325 293T-eGFPIBD 293T 293T- 293T- 6A Vandekerckhove, De Rijck et al, JVI 2006
Selection for resistance upon interfering with the interaction LEDGF/p75 L368 A128 E170 I365 D366 H171 INTEGRASE Hombrouck et al., Plos Pathogens 2007
Transportin-SR2 a binding partner of HIV-IN Christ et al., Current Biology,2008
MOA: Decreased nuclear import of PICs in HeLaP4 TRN-SR2 Knockdown Cells Christ et al., Current Biology, 1192–1202, August 26, 2008
Christ et al., Current Biology, 2008
Key resistance mutations reported Key resistance mutations reported for raltegravir/elvitegravir for raltegravir/elvitegravir E1 38A/G1 40A/G1 48K E92Q/N1 55H G1 40S/Q1 48R G1 40S/Q1 48H T66I/S1 53Y Elvitegravir F1 21 Y Raltegravir Q1 48K Q1 48R N1 55S N1 55H 1 10 100 1000 10000 Vandekerckhove et al., Reviews in Antiviral Therapy 2008
IN a 32kD enzyme IN a 32kD enzyme 288 1 N-terminal 50 CCD: DDE 112 C-terminal 288
Structural knowledge of HIV-1 Integrase NTD-NTD CCD-CCD CTD-CTD CCD-CTD dimer NTD-CCD tetramer full-length monomer
Information of the Tn5-transposase
Strand Transfer inhibitors S-1360 L-870,810 GS-9137 L-708,906 MK-0518 MK-2048
Enzymatic reactions of HIV-1 integrase Stage A The catalytic triad DD35E coordinates one Mg 2+ between the D64 and D116 . Stage B A secondary Mg 2+ is brought in the site upon binding of the viral cDNA. Stage C The 2 terminal nucleotides at 3’ end are cleaved. (3’ processing step). Stage D The free hydroxyl radical will attack a phosphodiester bond of the human DNA (strand transfer step). Stage E Stage X DKA are inhibitors binding after the 3’ The viral DNA is linked in the processing step by coordinating the dimetal motif in the site human DNA. and thereby competing with the binding of the human DNA, resulting in inhibition of the strand transfer step.
Proposed different mechanisms of resistance. Vandekerckhove et al, CROI 2009
Modeling of MK-O518 in integrase-3’-proc- cDNA complex Q148K/R/H Q146K Y143R/C G140S/A N155H E138 T66I T97A E92Q
Conclusions Conclusions LEDGF is a validated new target for ART LEDGF is a validated new target for ART Structural data are needed for further validation Structural data are needed for further validation of Transportin SRII of Transportin SRII Resistance pathway analysis of DKA based pathway analysis of DKA based Resistance integrase inhibitors allows: inhibitors allows: integrase – to give insight into the structure of the to give insight into the structure of the – functional IN complex functional IN complex – to propose different mechanisms of resistance to propose different mechanisms of resistance –
ACKNOWLEDGMENTS ACKNOWLEDGMENTS This work was possible with the support of This work was possible with the support of Arnout Voet Arnout Voet Chris Verhofstede Verhofstede Chris Zeger Debyser Zeger Debyser Dirk Vogelaers Vogelaers Dirk Daria Hazuda Daria Hazuda
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