Affinities between the Binding Partners of the HIV-1 Integrase Dimer-Lens Epithelium-derived Growth Factor (IN Dimer-LEDGF) Complex

Abstract

The interaction between lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF) and human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for HIV-1 replication. Homogeneous time-resolved fluorescence resonance energy transfer assays were developed to characterize HIV-1 integrase dimerization and the interaction between LEDGF and IN dimers. Using these assays in an equilibrium end point dose-response format with mathematical modeling, we determined the dissociation constants of IN dimers (K dimer ‫؍‬ 67.8 pM) and of LEDGF from IN dimers (K d ‫؍‬ 10.9 nM). When used in a kinetic format, the assays allowed the determination of the on-and off-rate constants for these same interactions. Targeting the viral integration process with small molecules as a strategy to inhibit HIV-1 2 replication has recently yielded an important new class of antiviral drugs. One integrase strand transfer inhibitor (INSTI), raltegravir (MK-0518), has been approved by the Food and Drug Administration for treatment of HIV-1 infection, and a second drug, elvitegravir (GS-9137), is in late stage clinical development. Based on binding experiments (1) and molecular modeling (2), strand transfer inhibitors are thought to interact with a pocket in the active site of integrase that is formed after 3Ј-processing of the viral DNA ends. INSTIs thus prevent the integrase-viral DNA complex from engaging host target DNA. More recently, a second site on integrase that represents the binding site for the cellular cofactor LEDGF was demonstrated to be a viable and attractive target for antiviral drug discovery (3-5). A small molecule inhibitor targeting this second site may retain activity against viral mutants resistant to INSTIs and complement the antiviral activity of INSTIs, akin to non-nucleoside reverse transcriptase inhibitors with nucleoside reverse transcriptase inhibitors. Hence, compounds that interact with the LEDGF binding pocket on IN could be used in combination with INSTIs to decrease the likelihood of resistance emergence.The cellular cofactor LEDGF has been identified as the dominant binding partner of HIV-1 integrase in human cells (6 -10). LEDGF interacts with integrase primarily through an ϳ80-amino acid domain termed the integrase binding domain (IBD) (11). A solution structure of the IBD has been derived (12). In addition, several co-crystal structures involving IBD and IN domains have been solved and include the following: IBD bound to a dimer of HIV-1 integrase catalytic core domain (CCD), IBD bound to an HIV-2 two-domain integrase (N-terminal domain (NTD) and catalytic core domain), and IBD bound to a maedi-visna virus two-domain (NTD ϩ CCD) integrase (13-15). Several lines of evidence point to the requirement of LEDGF for HIV-1 replication as a viral cofactor as follows. 1) Mutations in integrase that preserve the catalytic activity of the enzyme but cause defects in viral replication also disrupt integrase interaction with LEDGF (16 -18). 2) Suppression of LEDGF expression mediated by smal...

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif

“…While the high affinity of HIV IN for LEDGF/p75 has been documented previously 44 , our data provide a structure-based explanation. In comparison with the cellular partners, binding to HIV IN buries a considerably larger hydrophobic surface area.…”

Development of an AlphaScreen-Based HIV-1 Integrase Dimerization Assay for Discovery of Novel Allosteric Inhibitors

“…However, under the assay conditions we have chosen (the presence of detergent, Mg ] as low as 30 nM), their contribution should be negligible, even in the presence of a stimulator. 17,18 As a consequence, this assay, in its current state, is not suited for the discovery of specific IN tetramerization inhibitors. On the other hand, all IN dimerization inhibitors should also inhibit the formation of the functional IN tetramer (a dimer of dimers) and perhaps of higher order oligomeric species.…”

“…The first incubation step of the mixture is critical in that it allows time for monomer exchange between these dimers and thus formation of the signal-inducing, doubletagged heterodimers. An estimated half-life of ±87 min for the IN dimer was determined by Tsiang et al 17 in their HTRF-based assay. However, we could not simply extrapolate this value to our setup, as conditions are far from identical.…”

“…13,16,[20][21][22] Recently, a homogeneous time-resolved fluorescencebased (HTRF) assay measuring IN dimerization was reported and used in combination with mathematical modeling to study the dynamics of IN-IN and IN-LEDGF/p75 interactions. 17,18 This assay, however, to our knowledge, has not been validated for high-throughput screening (HTS) or used for the discovery of small-molecule inhibitors of IN. Hence, aforementioned studies and the inclination toward allosteric IN inhibitors encouraged us to develop an AlphaScreenbased IN dimerization assay suited for these purposes (outlined in Fig.…”

“…14 Second, molecules have been identified that bind across the IN dimer interface, establishing contacts with both monomers. [16][17][18][19] Consequently, these compounds stimulate dimerization and shift the oligomerization equilibrium to certain species, resulting in a loss of catalytic activity. 13,16,[20][21][22] Recently, a homogeneous time-resolved fluorescencebased (HTRF) assay measuring IN dimerization was reported and used in combination with mathematical modeling to study the dynamics of IN-IN and IN-LEDGF/p75 interactions.…”