Vicente Tur

DR4-selective tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) variants obtained by structure-based design

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent 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. Different tumor types respond either to DR4 or to DR5 activation, and chemotherapeutic drugs can increase the expression of DR4 or DR5 in cancer cells. Thus, DR4 or DR5 receptor-specific TRAIL variants would permit new and tumor-selective therapies. Previous success in generating a DR5-selective TRAIL mutant using computer-assisted protein design prompted us to make a DR4-selective TRAIL variant. Technically, the design of DR4 receptor-selective TRAIL variants is considerably more challenging compared with DR5 receptor-selective variants, because of the lack of a crystal structure of the TRAIL-DR4 complex. A single amino acid substitution of Asp at residue position 218 of TRAIL to His or Tyr was predicted to have a favorable effect on DR4 binding specificity. Surface plasmon resonance-based receptor binding tests showed a lowered DR5 affinity in concert with increased DR4 specificity for the designed variants, D218H and D218Y. Binding to DcR1, DcR2, and osteoprotegerin was also decreased. Cell line assays confirmed that the variants could not induce apoptosis in DR5-responsive Jurkat and A2780 cells but were able to induce apoptosis in DR4-responsive EM-2 and ML-1 cells.

Reconstruction of protein backbones from the brix collection of canonical protein fragments

As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more than 1,000 frequently observed conformations per peptide length for 6 different variation levels. Analysis of the performance of BriX revealed an average structural coverage of protein structures of more than 99% within a root mean square distance (RMSD) of 1 Angstrom. Globally, we are able to reconstruct protein structures with an average accuracy of 0.48 Angstrom RMSD. As expected, regular structures are well covered, but, interestingly, many loop regions that appear irregular at first glance are also found to form a recurrent structural motif, albeit with lower frequency of occurrence than regular secondary structures. Larger loop regions could be completely reconstructed from smaller recurrent elements, between 4 and 8 residues long. Finally, we observed that a significant amount of short sequences tend to display strong structural ambiguity between alpha helix and extended conformations. When the sequence length increases, this so-called sequence plasticity is no longer observed, illustrating the context dependency of polypeptide structures.

Enhancement of Antitumor Properties of rhTRAIL by Affinity Increase toward Its Death Receptors

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent and selective inducer of apoptosis in various tumor types, raising enthusiasm for TRAIL as a potential anticancer agent. TRAIL-induced apoptosis is mediated by death receptors 4 (DR4) and DR5. The design of rhTRAIL variants either with improved affinity or selectivity toward one or both death-inducing receptors is thought to enhance the therapeutical potential of TRAIL. Here we demonstrate that a single amino acid mutation at the position of glycine 131 to lysine or arginine in wild-type rhTRAIL significantly improved the affinity of rhTRAIL toward its death receptors, with the highest affinity increase observed for the DR4 receptor. These variants were able to induce higher in vitro levels of apoptosis in cancer cells responsive to only DR4 or to both death receptors and could therefore increase the potential use of rhTRAIL as an anticancer therapeutic agent.