Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration.

Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.

AlphaScreen-based characterization of the bifunctional kinase/RNase IRE1alpha: a novel and atypical drug target.

Assay technologies that were originally developed for high-throughput screening (HTS) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (ADMET) of HTS. Here the authors investigated and characterized the biological properties of a novel target, IRE1alpha, a bifunctional kinase/RNase stress sensor of the endoplasmic reticulum (ER). They have developed a novel assay platform using the HTS technology AlphaScreen to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1alpha. They show in vitro that dimerization/oligomerization of the cytosolic domain of IRE1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mRNA. Using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using AlphaScreen were biologically relevant. Preliminary characterization of assay robustness indicates that both AlphaScreen assays should be useful in HTS for the identification of IRE1 activity modulators.