Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants

Abstract

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

The Design and Characterization of Receptor-selective APRIL Variants

“…The effect on the interaction energy with TACI and BCMA was calculated as the difference in interaction energy (⌬⌬G, in kcal/mol) between the interaction energy of the mutant and the wild-type amino acid, using the AnalyseComplex option. In the case of preformed trimers, such as APRIL, the AnalyseComplex option was set as before (22,23,26) to consider the monomer subunits comprising APRIL as a single molecule and therefore did not discriminate in terms of binding energy on which particular monomer subunit a residue that interacts with the receptor is located.…”

“…Computational design of receptor selective mutants was performed as described previously (22,23,26). In short, amino acid residues with Van der Waals clashes, with bad torsion angles, or with a high energy in the crystal structure were repaired by replacing the side chain conformations (rotamers) observed in the x-ray structure by lower energy rotamers; hydrogen bond networks were optimized using the Repair PDB option of the FoldX protein design algorithm (19,27).…”

“…Due to the 3-fold symmetry of the APRIL-receptor complex, a "design unit" consisting of only two adjacent APRIL monomer subunits and a single receptor monomer subunit was used in the design process, similar to the one used in previous TRAIL design work (22,23,26). We used such a design unit for APRIL despite the fact that the receptor-binding interface of APRIL, in contrast to TRAIL, is largely confined to a single monomer subunit.…”

“…We used the FoldX protein design algorithm (19,20) to predict amino acid substitutions in APRIL that might switch ligand binding toward one or the other receptor. Previously, FoldX was successfully used to engineer variants of TRAIL that specifically recognize its receptor DR4 or DR5 (21)(22)(23)(24). Such a "branchpruning" approach has the added advantage that it allows selective removal of a single interaction, which contrasts with knock-out or knock-down studies that remove all interactions in which the target protein is involved (25).…”

A novel classification system to predict the pathogenic effects of CHD7 missense variants in CHARGE syndrome

“…A short combined steepest descent and simulated annealing minimization using constraints on aligned backbone atoms was performed, followed by a full unrestrained simulated annealing minimization for the entire model using the YASARA2 force field [Krieger et al, 2002;Krieger et al, 2004;Krieger et al, 2009]. Modeling of the CHD7 variants and the assessment of the effect on CHD7 stability was performed using the FoldX protein design algorithm [Guerois et al, 2002;Schymkowitz et al, 2005;Reis et al, 2010] as described previously [Alibes et al, 2010;Pey et al, 2007;Rakoczy et al, 2011;van der Sloot et al, 2006].…”