Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. The occurrence of DR5-responsive tumor cells indicates that a DR5 receptor-specific TRAIL variant will permit tumor-selective therapies. By using the automatic design algorithm FOLD-X, we successfully generated DR5-selective TRAIL variants. These variants do not induce apoptosis in DR4-responsive cell lines but show a large increase in biological activity in DR5-responsive cancer cell lines. Even wild-type TRAIL-insensitive ovarian cancer cell lines could be brought into apoptosis. In addition, our results demonstrate that there is no requirement for antibody-mediated cross-linking or membrane-bound TRAIL to induce apoptosis through DR5.computational protein design ͉ receptor selectivity ͉ biopharmaceutical ͉ death receptor ͉ apoptosis-inducing ligand 2 T umor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is currently attracting great interest as a potential anticancer therapeutic. TRAIL, in its soluble form, selectively induces apoptosis in tumor cells in vitro and in vivo by a death receptor-mediated process. Unlike other apoptosis-inducing TNF family members, soluble TRAIL appears to be inactive against normal healthy tissue (1). Reports in which TRAIL induces apoptosis in normal cells could be attributed to the specific preparations of TRAIL used (2). TRAIL shows a high degree of promiscuity as it binds to five cognate receptors: DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and the decoy receptors DcR1 (TRAIL-R3), DcR2 (TRAIL-R4), and osteoprotegerin (OPG) (3). Upon binding to TRAIL, DR4 and DR5 receptors recruit Fas-associated death domain, which binds and activates the initiator caspase 8, leading to apoptosis (4-6). DcR1 or DcR2 do not contain a death domain or a truncated death domain, respectively, and therefore could prevent apoptosis by sequestering available TRAIL or by interfering in the formation of a TRAIL-DR4 or -DR5 signaling complex (7).Use of TRAIL receptor-selective variants could permit better tumor-specific therapies through escape from the decoy receptor-mediated antagonism, resulting in a lower administrated dose with possibly fewer side effects and as alternatives to existing agonistic receptor antibodies (8-10). In experimental anticancer treatments, the receptors DR4 and͞or DR5 were shown to be up-regulated after treatment with DNA-damaging chemotherapeutic drugs, and the response to TRAIL-induced apoptosis was significantly increased (3, 11). In addition, irradiation appears to specifically up-regulate DR5 receptor expression, and the combination of irradiation and TRAIL treatment has been demonstrated to have an additive or synergistic effect (12). Thus, we chose to develop DR5 receptor-selective TRAIL variants by using a computational design strategy. Computatio...

Histone deacetylase inhibitors synergistically potentiate death receptor 4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cells

“…1, 3), suggesting that other factors regulate the sensitivity at the downstream signaling of the receptors. However, it seems that some leukemia cells undergo apoptotic cell death through signaling by either pro-apoptotic DR4 or DR5, but not both, as has been found for various solid tumors [25,26]. The substantial sensitivity of HL-60 (AML) and U937 (monoblastic leukemia) cells to TRAIL-mediated cytotoxicity, but not to AY4, suggests that DR5 is the main receptor that transduces apoptotic signaling in the leukemia cells.…”

“…The receptor-specific agonistic mAb studies demonstrated that, while various solid tumors were more sensitive to DR5-mediated apoptosis than DR4-induced apoptosis [25,26], primary CLL cells underwent apoptotic cell death almost exclusively through DR4, not DR5 [23,24,27]. However, there have been limited studies of how DR4-or DR5-mediated signaling alone or in combination with chemotherapeutic agents can contribute to apoptosis for other leukemia cells, such as ALL, AML, and CML cells.…”

Increased Apoptosis Induction in Hepatocellular Carcinoma by a Novel Tumor-Targeted TRAIL Fusion Protein Combined With Bortezomib

“…The enhanced activity of targeted TRAIL might be explained by its action as a quasi membrane-bound form, leading to efficient formation of both TRAIL-R1-and TRAIL-R2-signaling complexes, whereas scTRAIL preferentially binds to TRAIL-R1 and TRAIL decoy receptors. 42 Despite similar expression of both receptors in certain cancer cells, TRAIL-R2 appears as the primary transducer of the TRAIL death signal in many cancer cells, 43 which might account for the higher proapoptotic activity of EGFR-targeted scTRAIL.…”

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

“…In order to identify and characterize the critical residues involved in dimerization of ZFNs, 3D protein models based on the crystal structure of the native FokI endonuclease 22 25 were established. Although the published crystal structures 22,23 do not provide a clear-cut view of the active enzyme bound to DNA, they offer useful information for structure-aided design of point mutations in order to alter the dimerization behavior of ZFNs (Fig.…”