Résumé
Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 mu s replica-exchange molecular dynamics simulations to investigate some of these mutationsG85R/G86R (GGRR(mut)), Q117G (QG(mut)), and G85R/G86R/Q117G (GGRR/QG(mut)). GGRR(mut) turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QG(mut) optimizes them. Combining QG(mut) with GGRR(mut) compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRR(mut)-based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QG(mut)-based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QG(mut)-based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.