Christine Bruun Schiødt

The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue.

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, α-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.

Small‐Molecule Affinity Ligands for Protein Purification: Combined Computational Enrichment and Automated In‐line Screening of an Optically Encoded Library

As a result of their complexity, the purification of protein biopharmaceuticals poses challenges not encountered with traditional small-molecule drugs and requires lengthy series of chromatographic steps. Affinity chromatography (AC) is perhaps the most powerful chromatographic technique available, but is limited in scope because of the lack of suitable ligands for proteins for which no natural small-molecule binding partner or inhibitor exists. Current AC ligands used for targeting protein surfaces are primarily other proteins, such as a monoclonal antibody (mAb) or another binding protein. These ligands are expensive and amenable to rapid degradation under common column-cleaning conditions. Selective, chemically robust small-molecule ligands would greatly expand the scope and use of AC for the purification of biopharmaceuticals. However, small-molecule ligands targeting large protein surfaces are rare, despite significant recent achievements. The reason remains the inherent difficulty in designing small molecules that bind tightly and specifically to a large, featureless, amphiphilic, and flexible protein surface. In this study we have focused on identifying smallmolecule affinity ligands for human growth hormone (hGH) through the screening of a combinatorial library of compounds designed to mimic the interactions with its native receptor. Pioneering work has shown that the majority of the binding energy of the initial interaction between hGH and the extracellular part of its receptor (hGHbp) is conferred by a central functional epitope, or “hot spot”, dominated by two tryptophan residues (Trp104 and Trp169) in close proximity on the receptor (Figure 1). 5] This ensemble of residues essential for binding suggests that small-molecule binders may be designed to mimic the interactions of the biological protein partner.