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

Abstract

In recent years, HIV-1 integrase (IN) has become an established target in the field of antiretroviral drug discovery. However, its sole clinically approved inhibitor, the integrase strand transfer inhibitor (INSTI) raltegravir, has a surprisingly low genetic barrier for resistance. Furthermore, the only two other integrase inhibitors currently in advanced clinical trials, elvitegravir and dolutegravir, share its mechanism of action and certain resistance pathways. To maintain a range of treatment options, drug discovery efforts are now turning toward allosteric IN inhibitors, which should be devoid of cross-resistance with INSTIs. As IN requires a precise and dynamic equilibrium between several oligomeric species for its activities, the modulation of this equilibrium presents an interesting allosteric target. We report on the development, characterization, and validation of an AlphaScreen-based assay for high-throughput screening for modulators of HIV-1 IN dimerization. Compounds identified as hits in this assay proved to act as allosteric IN inhibitors. Additionally, the assay offers a flexible platform to study IN dimerization.

Small-Molecule Inhibitors of the LEDGF/p75 Binding Site of Integrase Block HIV Replication and Modulate Integrase Multimerization

“…His 6 -tagged HIV-1 integrase, glutathione S-transferase (GST)-tagged HIV-1, and 3ϫFlag-tagged LEDGF/p75 were purified for AlphaScreen applications as described previously (4,11,43).…”

The emergence of drug resistant HIV variants and novel anti–retroviral therapy

“…Most importantly, the sub-optimal regimens might have directed the course of HIV evolution dissimilar to that of the western societies. Nevertheless, in order to most efficiently combat the pandemic in different regions of the world, any attempt toward novel drug discovery should take such various evolutionary pathways into account [64] .…”

Fueling HIV-1 integrase drug design with structural insights

“…On the other hand, several groups have reported small molecules and peptides that modulate the IN oligomerization process in the opposite direction, shifting the equilibrium to the dimer or tetramer (reviewed in [11]). The best evidence for such a mechanism has been provided for peptides comprising LEDGF/p75 residues 361-370, LEDGINs and the largely symmetrical biscaffeoyl compound described above [23,40].…”

“…Other, purely allosteric inhibitors of IN have been reported as well and various starting points for in silico optimization are present in the literature [12][13][14]. A further strategy to inhibit IN activity is to perturb its crucial oligomerization equilibrium, either by shifting it to the inactive monomer or to a certain other species like the dimer or tetramer [11,22,23]. Also in this area, ample structural data are available to aid drug design.…”