Integrase

 

The human immunodeficiency virus 1 (HIV-1) is the causative agent for acquired immunodeficiency syndrome (AIDS) in humans. It is a retrovirus that uses ribonucleic acid (RNA) as its genetic material within each enveloped virus particle.  The virus uses this RNA to make a deoxyribonucleic acid (DNA) copy with the help of a nucleic acid synthesizing enzyme, reverse transcriptase, which creates a DNA copy of the virus genome. This DNA copy is subsequently inserted into the host cell’s genome, by yet another viral enzyme, integrase. The virus hereafter hijacks the cellular machinery to replicate itself along with the host cell DNA. These two critical steps, i.e. the synthesis of nucleic acid material and consequent integration into host cell DNA, are controlled by the virus and ensure the continued replication of the virus as long as that cell is alive.

 

The integrase enzyme has 3 functionally distinct domains: a zinc finger motif-containing ‘metal chelator’ domain at the N terminal, a central ‘catalytic core’ domain that performs a two-step integration process and a third variable C terminal ‘DNA binding’ domain. These three domains concertedly bring about the efficient creation of a provirus in the host cell [1].

 

 Target for drugs

An important premise of drug discovery research is the identification of several essential steps in the virus life cycle that can be used as targets for intervention. Two essential enzyme activities of HIV-1 replication i.e. reverse transcriptase and protease have been successfully targeted for drug discovery and development. Integrase is also crucial for the propagation of the virus and therefore an equally attractive target for therapeutic intervention. This function is unique to the virus because while the host cell makes many enzymes that carry out polymerase and protease activities, it does not produce or need integrase-like enzymes [1] for its normal cellular activities. Therefore the search for integrase inhibitors has been the subject of intense research in recent years.

 

 Integrase Inhibitors (InSTI)

Several InSTIs have been described in the literature [1]. These belong to a class of ‘catalytic inhibitors’ because they inhibit the catalytic ‘strand transfer’ activity of the enzyme. Early drug discovery research using high throughput screening technology identified a ‘b-diketo acid’ chemical skeleton as an effective inhibitor of integrase and consequently became a scaffold chemical structure for InSTIs that followed. The first InSTI raltegravir (RAL) developed by Merck, was approved in 2007. RAL was initially evaluated among treatment-experienced patients, but was recently also approved for use in treatment naïve patients [2]. Two RAL Phase 3 studies Benchmrk 1 & 2 demonstrated [3] that when used in combination with an optimized background regimen, the overall mean viral load declined from baseline by -1.77 log₁₀, in subjects who had evidence of resistance to 3 classes of antiretrovirals (ARVs).

 

The second member of InSTIs, elvitegravir (EVG) is currently in Phase 3 testing and is being developed by Gilead Sciences. Previous Phase 2 studies indicated that EVG was a potent ARV when used in combination with an optimized background regimen in patients having evidence of triple class resistance [1].  A third InSTI being developed jointly by Shinogi and GlaxoSmithKline, S/GSK1349572 was recently shown to be extremely potent at reducing the HIV-1 viral load in a 10 day monotherapy study [4], with the highest reduction of baseline viral load ever reported.

 

These inhibitors appear to have functional moieties that serve complementary functions: a flexible ‘enzyme binding moiety’ that helps stabilize the inhibitor binding to the enzyme and a ‘catalytic core moiety’ that has a metal chelating activity which helps in sequestering essential metal ions from the active site thus rendering the enzyme inactive [1].

 

Several reports describing in vitro and in vivo resistance to this class of inhibitors are available and naturally occurring polymorphisms in the integrase gene have also been documented [1]. Multiple pathways exist that lead to the development of resistance: clinical trials of RAL have identified the emergence of resistance associated mutations (RAMs), e.g. N155H in combination with any of L74M, E92Q, G163R as one pathway or Q148H/R/K with E138K or G140S/A as a second and more recently Y143R/C as a third pathway [5,6]; similarly, clinical trials of EVG have shown that subjects who had virological failure exhibited the mutations T66I/A/K and S147G in addition to the previously listed mutations E92Q, E138K, Q148H/R/K and N155H [7,8]. While other InSTIs have exhibited other RAMs, considerable overlap in invitro resistance profiles has been observed [1]. Most RAMs are clustered in the catalytic moiety of the enzyme and are believed to reduce susceptibility by interfering with the drug binding to the target region(s) of the enzyme.

 

An evaluation of polymorphic mutations in the integrase gene among InSTI-naïve subjects has revealed that the mutations V72I, L74I, T97A, V151I/L, E157Q, V165I, V201I, I203M, T206S and S230N did not significantly alter the susceptibility to InSTIs in the absence of RAMs, with the exception of E157Q [9].

 

Resistance to antiretroviral drugs remains an important limitation to successful HIV-1 therapy.

Drug resistance testing has become widespread in the developed world and has been accepted as an important adjunct to the management of patients with detectable plasma viremia who are receiving antiretroviral therapy.   Moreover, person-to-person transmission of drug-resistant viruses occurs in a variety of settings, including between adults and from mother to child, indicating that testing for drug resistance before initiating therapy may be useful even for treatment-naive patients.

 

The International AIDS Society–USA[i]EuroGuidelines Group for HIV resistance recommends that resistance testing be performed at the time of HIV infection diagnosis as part of the initial comprehensive patient assessment, as well as in all cases of virologic failure, whenever possible.   Tropism testing is recommended whenever the use of chemokine receptor 5 antagonists is contemplated.   Currently, commercially available assays to measure mutations associated with treatment resistance to integrase inhibitors are available from Monogram Biosciences (PhenoSense Integrase, Quest Diagnostics, Celera (ViroSeq) and Siemens Medical (TruGene).  [ii]

 

Virco BVBA has a research use only Integrase assay currently available, and will launch a quantitative resistance prediction from genotype (vircoTYPE INT) in 2010, in specific territories, dependent on technical and regulatory requirements.

 

1.       Jegede et. al. (2008). AIDS Reviews, 10: 172-189.

2.       US FDA Package insert for Isentress, Revision 2009.

3.       Steigbigel et. al. (2008) New Engl. J Med. 359: 339-354.

4.       Lalezari et. al. (2009). 5^th IAS Conference on Pathogenesis, Treatment & Prevention, Cape Town, S. Africa, Abstract TUAB105.

5.       Hazuda et. al. (2007). XVI International HIV Drug Resistance Workshop: Basic Principles and Clinical Implications, Bridgetown, Barbados, Abstract 8.

6.       Hatano et. Al. (2008). XVII International HIV Drug Resistance Workshop, Sitges, Spain, Abstract 10.

7.       Jones et. al. (2007). 14^th Conference on retroviruses and Opportunistic Infections, Los Angeles, USA, Abstract 627.

8.       McColl et al. (2007). XVI International HIV Drug Resistance Workshop: Basic Principles and Clinical Implications, Bridgetown, Barbados, Abstract 9.

9.       Low et. al. (2009). Antimicrob Agents & Chemotherapy, in press.