HIV- -1 Integrase: 1 Integrase: HIV not just an not just an - - PowerPoint PPT Presentation

HIV HIV-

• 1 Integrase:

1 Integrase: not just an not just an

• ther HIV enzyme
• ther HIV enzyme

Vandekerckhove Linos MD, PhD

• Dpt. of Infectious Diseases

University Hospital Ghent Belgium

IN a 32kD enzyme IN a 32kD enzyme

1 288 50 112 CCD: DDE N-terminal C-terminal 288

Vandekerckhove et al., Reviews in Antiviral Therapy 2008

Z T T T Viral DNA

2-LTR circles Integrated DNA

BLOCK (before) NI BLOCK after NI

T T T T T T

Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen

Brass et al., Science. 2008 Feb

HIV HIV-

• cofactors

cofactors

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 with the interaction (J. Coffin rule)

New ART target

LEDGF/p75, a novel LEDGF/p75, a novel Binding partner of HIV Binding partner of HIV-

• IN

IN Identified as binding partner of HIV Identified as binding partner of HIV-

• 1 IN

1 IN

( (Cherepanov Cherepanov et al., 2003; et al., 2003; Maertens Maertens et al., 2003) et al., 2003)

Lentiviral Lentiviral specific and increasing affinity of IN for specific and increasing affinity of IN for DNA DNA

( (Busschots Busschots et al., 2005) et al., 2005)

IBD

1 91 148 156 325 347 429 530 aa

PWWP

NLS AT

p52 p75

Q-PCR to pinpoint the replication block

Reverse transcription Integrated proviral DNA

10 20 30 40 50 8 24 48 72 Hours Post Infection DNA copies / cell

293T 293TeGFP 293T-eGFPIBD 293T-eGFPΔ325 293T-eGFPΔ325D366A

0,5 1 1,5 2 293T 293T- eGFPΔ325D36 6A 293T- eGFPΔ325 293T-eGFPIBD

Non-Integrated Nuclear Viral DNA

0,2 0,4 0,6 0,8 1 1,2 1,4 8 24 48 72 Hours Post Infection 2LTR Circles / Cell

293T 293TeGFP 293T-eGFPIBD 293T-eGFPΔ325 293T- eGFPΔ325D366A

Integrated copies/cell

Vandekerckhove, De Rijck et al, JVI 2006

Selection for resistance upon interfering with the interaction

D366 H171 E170 LEDGF/p75 INTEGRASE A128 I365 L368

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

1 10 100 1000 10000

N1 55H N1 55S Q1 48R Q1 48K F1 21 Y T66I/S1 53Y G1 40S/Q1 48H G1 40S/Q1 48R E92Q/N1 55H E1 38A/G1 40A/G1 48K

Elvitegravir Raltegravir

Vandekerckhove et al., Reviews in Antiviral Therapy 2008

IN a 32kD enzyme IN a 32kD enzyme

1 288 50 112 CCD: DDE N-terminal C-terminal 288

Structural knowledge of HIV-1 Integrase

NTD-NTD CCD-CCD CTD-CTD NTD-CCD tetramer CCD-CTD dimer 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

• ne Mg2+ between the D64 and D116.

Stage B

A secondary Mg2+ 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

The viral DNA is linked in the human DNA.

Stage X DKA are inhibitors binding after the 3’

processing step by coordinating the dimetal motif in the site 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

E92Q T97A E138 G140S/A

Q148K/R/H

N155H Y143R/C Q146K T66I

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

• f Transportin SRII
• f Transportin SRII

Resistance Resistance pathway analysis of DKA based pathway analysis of DKA based integrase integrase inhibitors allows: inhibitors allows: – – 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 Arnout Voet Voet Chris Chris Verhofstede Verhofstede Zeger Zeger Debyser Debyser Dirk Dirk Vogelaers Vogelaers Daria Daria Hazuda Hazuda