International Conference on Retroviral Integration edition:4 location:Siena, Italy date:4-7 October 2011
DEVELOPMENT OF AN ALPHASCREEN®-BASED HIV-1 INTEGRASE DIMERIZATION ASSAY LEADING TO THE DISCOVERY OF NOVEL SMALL MOLECULE INHIBITORS
Jonas Demeulemeester*, Zeger Debyser, Frauke Christ
Molecular Medicine, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium
* E-mail: firstname.lastname@example.org
Background. Human Immunodeficiency Virus-1 Integrase (HIV-1 IN) inserts the viral DNA into host cell chromatin in a multistep process. This enzyme exists in equilibrium between monomeric, dimeric, tetrameric and high order multimeric states. However, monomers of IN are not capable of supporting its catalytic functions and the active forms have been shown to be a dimer for 3’ processing and even a tetramer for strand transfer.
Aims. As a consequence of the strict correlation between catalytic activity and specific oligomeric forms of the IN, the development of modulators of the IN oligomerization equilibrium constitutes a promising novel antiviral strategy with proof of principle in the literature.
Results. In this work, we set up, characterized and validated an IN dimerization assay using the AlphaScreen® technology platform. Our assay is able to detect both inhibitors and stimulators of IN dimerization and is suited for high-throughput screening (Z’(inhib) = 0.77 & Z’(stim) = 0.59). We also implemented a GST-His6 AlphaScreen® counterscreen to rid hit lists of quenching false positives. Our screening pipeline was used to evaluate molecules emerging from an in silico drug discovery campaign, resulting in several small molecule inhibitors of IN dimerization (Tintori et al. University of Siena, Italy). The most active compound also inhibited overall IN catalytic activity in an ELISA format. Additionally, the assay was applied to evaluate IN dimerization mutants and is currently being used in a high-throughput screening campaign (Serrao et al. University of Southern California, LA, CA, USA).
The development of this assay is already leading to the discovery of novel allosteric IN inhibitors. Some of these may ultimately be optimized to overcome resistance to the traditional IN strand transfer inhibitors.