Basic Science of Integrase Inhibitors

Mark A Wainberg McGill University AIDS Centre Montreal, Canada I have no conflicts of interest to disclose. HIV Replication Cycle and Drug Targets a. Entry inhibitors b. Reverse transcriptase inhibitors c. Protease inhibitors d. 3′-processing inhibitors e. Strand transfer inhibitors

Pommier Y, et al. Nat Rev Drug Discov. 2005;4:236-248. The Role of Integrase Inhibitors in HIV Management

Integrase Enzyme

. Viral enzyme essential to replication of both HIV-1 and HIV-2 . Integration – Follows reverse transcription, where DNA copy of HIV-1 RNA synthesized after infection – Essential step before viral DNA can be transcribed back into viral RNA – Incorporates or “integrates” viral DNA into host cell’s DNA

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3 Domains of Integrase Enzyme

. Integrase enzyme: 32-kDa protein encoded by integrase portion of the viral pol gene . 3 domains defined by x-ray crystallography or nuclear magnetic resonance imaging – N-terminal zinc finger domain enhances multimerization and promotes integration of the 2 ends of viral DNA into host cell chromosome – Dimeric central catalytic domain required for enzymatic activity of integrase – C-terminal domain directs metal- and sequence-independent DNA binding . Current integrase inhibitors disrupt by binding to catalytic domain

Semenova E, et al. Adv Pharmacol. 2008;56:199-228. clinicaloptions.com/hiv The Role of Integrase Inhibitors in HIV Management

4 Key Steps in Integration

. Assembly of a stable PIC after reverse transcription of viral DNA . 3′-end processing . Strand transfer . Creation of intact double-stranded DNA

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3′-Processing and Strand Transfer

. 3′-processing occurs in host-cell cytoplasm after reverse transcription . Strand transfer inserts both viral DNA ends into host-cell chromosome (blue) . Each of the 3′-ends of the viral DNA (red circles) attacks a DNA bond on each strand of host DNA with a 5-base-pair stagger across DNA major groove (d), leaving – Single-stranded gap at each junction between integrated viral DNA and host DNA – 2-base flap at 5′-ends of viral DNA (d and e) . Gap filling and release of unpaired 5′-ends of viral DNA (arrows in e) carried out in coordination with cellular repair enzymes

Pommier Y, et al. Nat Rev Drug Discov. 2005;4:236-248. clinicaloptions.com/hiv Integration

3’-processing strand transfer

Metifiot, Viruses, 2010 The Role of Integrase Inhibitors in HIV Management Mechanisms of Action of Integrase Inhibitors . Potential targets for small-molecule integrase inhibitors – Integrase enzyme before substrate binding – Viral DNA substrate – PIC . Most development efforts focused on 2 mechanisms – Inhibition of PIC assembly – Inhibition of strand transfer

clinicaloptions.com/hiv The Role of Integrase Inhibitors in HIV Management Possible Mechanisms of Integrase Inhibitor Resistance . Reduced interaction of inhibitor with divalent metal ions in active site[1] . Reduced interaction of inhibitor with enzyme amino acids in hydrophobic binding pockets that are not in active site but affect ability of inhibitor and enzyme to interact[2,3] . To date, resistance-conferring mutations to raltegravir and elvitegravir appear to involve the first mechanism . Genotypic and phenotypic assays to detect and measure resistance in development

1. Hazuda DJ. CCO HIV Annual Update 2007. 2. Hazuda DJ, et al. Proc Natl Acad Sci U S A. 2004;101:11233-11238. 3. Savarino A. Retrovirology. 2007;4:21. clinicaloptions.com/hiv The Role of Integrase Inhibitors in HIV Management Mutations Resistant to 3 Types of InIs Surround Metal Ions in Active Site . Highly conserved residues; low potential for preexisting resistance

Q148 Catalytic residues S153 Diketoacid Naphthyridine G140 E138 Pyrimidine N155

T66 F121 Mg

V72 T125

Adapted with permission of Merck & Co., Inc., Whitehouse Station, NJ. Copyright © Merck & Co., Inc. Whitehouse Station, NJ. All rights reserved. clinicaloptions.com/hiv The Evolving Role of Integrase Inhibitors in HIV Therapy clinicaloptions.com/hiv

HIV Viral Life Cycle

Maturation

Budding Uncoating Attachment Integration fusion

Reverse Assembly transcription Transcription, translation The Evolving Role of Integrase Inhibitors in HIV Therapy clinicaloptions.com/hiv

HIV Viral Life Cycle

Currently available integrase inhibitors . Raltegravir (approved 10/07) . Elvitegravir* (approved 8/12) . Dolutegravir (approved 8/13) *Currently available only as part of a coformulated single-tablet regimen. Maturation

Uncoating Budding Attachment Integrase fusion inhibitors

Reverse Assembly transcription Transcription, translation

No compensatory mutations in regard to DTG resistance and viral fitness have developed over more than five years in culture. Acknowledgements • Bluma Brenner • Hongtao Xu • Jerry Zaharatos • Maureen Oliveira • Thibault Mesplède • Peter Quashie • Kaitlin Anstett • Vincent Cutillas • Said Hassounah • Nathan Osman • Agnès Depatureaux