Inactivation of TNF signaling by rationally designed dominant-negative TNF variants.

Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.

Computational stabilization of human growth hormone.

Recombinant human growth hormone (hGH) is used worldwide for the treatment of pediatric hypopituitary dwarfism and in children suffering from low levels of hGH. It has limited stability in solution, and because of poor oral absorption, is administered by injection, typically several times a week. Development has therefore focused on more stable or sustained-release formulations and alternatives to injectable delivery that would increase bioavailability and make it easier for patients to use. We redesigned hGH computationally to improve its thermostability. A more stable variant of hGH could have improved pharmacokinetics or enhanced shelf-life, or be more amenable to use in alternate delivery systems and formulations. The computational design was performed using a previously developed combinatorial optimization algorithm based on the dead-end elimination theorem. The algorithm uses an empirical free energy function for scoring designed sequences. This function was augmented with a term that accounts for the loss of backbone and side-chain conformational entropy. The weighting factors for this term, the electrostatic interaction term, and the polar hydrogen burial term were optimized by minimizing the number of mutations designed by the algorithm relative to wild-type. Forty-five residues in the core of the protein were selected for optimization with the modified potential function. The proteins designed using the developed scoring function contained six to 10 mutations, showed enhancement in the melting temperature of up to 16 degrees C, and were biologically active in cell proliferation studies. These results show the utility of our free energy function in automated protein design.