Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations

Abstract

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 Angbased 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 ms replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/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.

CSPG4: A Target for Selective Delivery of Human Cytolytic Fusion Proteins and TRAIL

“…Enzymatic activity may be influenced via various methods involving modification of the cytolytic effector peptide in order to reduce its sensitivity to endogenous inhibitors and/or boost its affinity for catalytic substrates [ 73 , 74 ]. Granzyme B (R201K) [ 44 , 57 ] and angiogenin (G85R/G86R and Q117G) [ 39 , 40 ] are examples pro-apoptotic proteins which have undergone such modifications to enhance their cytolytic activity.…”

Critical Issues in the Development of Immunotoxins for Anticancer Therapy

“…92 To prevent the hypersensitivity and intrinsic immunogenicity associated with nonhuman toxins, human endogenous cytotoxic proteins have been utilized as an alternative payload in fourth-generation ITs. 21,93 Such ITs have been generated based on GrB, 94,95 angiogenin, 96 pancreatic RNase A, 8 microtubule-associated protein tau, 97 and deathassociated protein kinase 98 and have shown in vitro and in vivo antitumor activities. Nonetheless, the cytotoxicity of human enzymatic toxins is compromised by endogenous inhibitors in the cytosol: GrB is suppressed by serine protease inhibitor B9 (serpin B9 or PI9), 22 and pancreatic RNase A by the ribonuclease inhibitor (RI) protein.…”

Advances in epidermal growth factor receptor specific immunotherapy: lessons to be learned from armed antibodies

“…Using its nuclear ribonuclease activity, Ang has shown to primarily function to regulate angiogenesis and positively influence the activation of molecular pathways driving cancer's metastatic, invasive and migratory potential [33,36,211]. Also, Ang has shown the ability to translocate in the cell cytosol in response to oxidative stress and induce apoptosis by abolishing protein synthesis through tRNA, 5S, 18S, and 28S rRNA hydrolysis [34,36,223,224]. Unfortunately, Ang therapeutic efficacy has been hampered by the antagonistic effect of the endogenous human placental ribonuclease inhibitor 1 (RNH1), which acts to prevent self-tissue damage [34,212,225,226].…”

“…Also, Ang has shown the ability to translocate in the cell cytosol in response to oxidative stress and induce apoptosis by abolishing protein synthesis through tRNA, 5S, 18S, and 28S rRNA hydrolysis [34,36,223,224]. Unfortunately, Ang therapeutic efficacy has been hampered by the antagonistic effect of the endogenous human placental ribonuclease inhibitor 1 (RNH1), which acts to prevent self-tissue damage [34,212,225,226]. To bypass this obstacle, Cremer et al (2015) and Gresch et al (2018), engineered multiple Ang mutant versions, which have decreased affinity for their RNH1 [34,223].…”