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 HIV and Coverage of the
17th Conference on Retroviruses and
Infections (CROI 2010)
 February 16 - 19, San Franciso, California
LEDGF/p75 Integrase Inhibitors and Capsid Assembly Inhibitors Offer New Approaches for Blocking HIV Replication

SUMMARY: While the drug development pipeline is not as full as it has been in recent years, researchers continue to work on new approaches to antiretroviral therapy. Two such novel approaches -- LEDGF integrase inhibitors and capsid assembly inhibitors -- were described in oral presentations at the recent 17th Conference on Retroviruses and Opportunistic Infections (CROI 2010) last month in San Francisco.

By Liz Highleyman

In order to replicate, HIV must enter a host cell, shed its outer coat, copy its genetic material (transcribing RNA into DNA, the job of reverse transcriptase), and insert the new copy into the host cell's chromosome (the job of integrase). The virus then "hijacks" the cell's machinery to produce large viral proteins, which are cut up (the job of protease) and assembled into new viral particles.

LEDGF Inhibitors

Frauke Christ from Catholic University in Leuven, Belgium, presented data on a new type of HIV integrase inhibitor that interferes with a cellular protein known as LEDGF (lens epithelium-derived growth factor, or p75). Unlike the approved integrase inhibitor raltegravir (Isentress), this approach does not interfere with the integrase enzyme directly, but rather focuses on its "binding partner," LEDGF/p75.

In 2003, the investigators identified LEDGF/p75 as a strong integrase binding partner, and they have since established a validation procedure for novel HIV cofactors. This allowed them to confirm the importance of LEDGF/p75 -- which tethers HIV integrase to the cellular genome -- in the process of HIV replication.

Understanding another step in the HIV lifecycle, of course, offers a new drug target. X-ray crystallography of the integrase-binding domain revealed that small molecule protein-protein interaction inhibitors could potentially disrupt the LEDGF/p75-integrase interaction.

The researchers used rational drug design to identify small molecules likely to fit the interaction site, narrowing their search to a set of compounds known as 2-(quinolin-3-yl) acetic acid derivatives (dubbed LEDGINs). From an initial 2 dozen candidates, they found one (CX00287) that moderately inhibited LEDGF/p75-integrase interaction and HIV replication in vitro.

They tinkered further with the shape of most promising candidate, creating optimized versions. The most promising, CX04328, was a potent inhibitor of LEDGF/p75-integrase interaction and HIV replication -- but not of other cellular functions -- in laboratory studies. It showed no toxicity in cell cultures and was not cross-resistant with strand-transfer integrase inhibitors such as raltegravir and the experimental integrase inhibitor elvitegravir.

The selectivity of the chosen compound was considered inadequate, so work continued, yielding a modified compound -- CX06387 -- that was the most potent so far, was highly selectivity, and produced good preliminary toxicity data, making it a promising candidate for further clinical development. Christ said initial animal studies are underway and projected that these might be finished in about a year.

"Our work demonstrates the feasibility of rational design of small molecules inhibiting the protein-protein interaction between a viral protein and a cellular host factor," the investigators concluded. The discovery of the 2-(quinolin-3-yl) acetic acid derivatives as the first agents to inhibit the LEDGF/p75-integrase interaction and HIV replication "provides the ultimate proof for the crucial role of the co-factor in HIV replication."

Capsid Assembly Inhibitors

Steve Titolo from Boehringer Ingelheim Canada and colleagues described the development of novel capsid assembly inhibitors, which prevent construction of the cone-shaped shell surrounding newly produced viral genetic material -- a crucial step in forming functional new viral particles. There are currently no approved assembly inhibitor dugs.

The investigators developed an in vitro capsid assembly assay, which they used to screen libraries of potential compounds. Inhibition of wild-type and drug- resistant virus was studied in C8166 T-cells. Binding to the capsid was determined using nuclear magnetic resonance spectroscopy and X-ray crystallography.

Screening yielded several different clusters of structurally related compounds (dubbed chemotypes) that inhibited capsid assembly. The researchers optimized 2 promising chemotypes -- benzodiazepines and benzimidazoles -- producing compounds with potent antiviral activity against both wild-type HIV and virus resistant to existing antiretroviral drug classes.

The two chemotypes both reduced production of infectious virions (virus particles), but they had different effects, with benzodiazepines preventing formation of mature virions and benzimidazoles causing production of progeny virus with profound core morphology defects.

Structural analysis showed that both selected chemotypes bound to the N-terminal domain of capsid by forming a binding pocket, which overlaps with the binding site for a previously reported type of capsid assembly inhibitors. However, the 2 chemotypes had different shapes, with the benzimidazoles protruding more from the binding pocket.

Letting the virus replicate in the presence of the new inhibitors led to emergence of several resistance mutations, mostly in highly conserved regions in or near the binding pocket; different sets of resistance mutations were selected by the 2 chemotypes, but there was some cross-resistance. Most of the resistance mutations impaired viral replicative capacity.

Development of the 2 specific lead compounds was discontinued due to potency issues, which Titolo said was probably due to their lipophilic nature.

Nevertheless, the researchers concluded that their work "demonstrated a proof of concept" that potent capsid inhibitors could be developed as new anti-HIV drugs.

Study 1: Katholieke Univ Leuven, Belgium; Center for Innovation and Stimulation of Medical Development, Leuven, Belgium.

Study 2: Boehringer-Ingelheim Ltd, Laval, Canada; University of Utah, Salt Lake City, UT.



F Christ, A Voet, A Marchand, and others. First-in-class Inhibitors of LEDGF/p75-integrase Interaction and HIV replication. 17th Conference on Retroviruses & Opportunistic Infections (CROI 2010). San Francisco. February 16-19, 2010. Abstract 49.

S Titolo, J-F Mercie, E Wardrop, and others. Discovery of Potent HIV-1 Capsid Assembly Inhibitors. 17th Conference on Retroviruses & Opportunistic Infections (CROI 2010). San Francisco. February 16-19, 2010. Abstract 50.



















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