Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening

Abstract

Granulocyte-colony stimulating factor (G-CSF) is used worldwide to prevent neutropenia caused by highdose chemotherapy. It has limited stability, strict formulation and storage requirements, and because of poor oral absorption must be administered by injection (typically daily). Thus, there is significant interest in developing analogs with improved pharmacological properties. We used our ultrahigh throughput computational screening method to improve the physicochemical characteristics of G-CSF. Improving these properties can make a molecule more robust, enhance its shelf life, or make it more amenable to alternate delivery systems and formulations. It can also affect clinically important features such as pharmacokinetics. Residues in the buried core were selected for optimization to minimize changes to the surface, thereby maintaining the active site and limiting the designed protein's potential for antigenicity. Using a structure that was homology modeled from bovine G-CSF, core designs of 25-34 residues were completed, corresponding to 10 21 -10 28 sequences screened. The optimal sequence from each design was selected for biophysical characterization and experimental testing; each had 10-14 mutations. The designed proteins showed enhanced thermal stabilities of up to 13°C, displayed five-to 10-fold improvements in shelf life, and were biologically active in cell proliferation assays and in a neutropenic mouse model. Pharmacokinetic studies in monkeys showed that subcutaneous injection of the designed analogs results in greater systemic exposure, probably attributable to improved absorption from the subcutaneous compartment. These results show that our computational method can be used to develop improved pharmaceuticals and illustrate its utility as a powerful protein design tool.

Rational Design of Intercellular Adhesion Molecule-1 (ICAM-1) Variants for Antagonizing Integrin Lymphocyte Function-associated Antigen-1-dependent Adhesion

“…One of the variant libraries was designed using combined output from Protein Design Automation ® (PDA) (13,17,18) and Sequence Prediction Algorithm ™ (SPA) (19) calculations. For PDA calculations, the conformations of amino acids at variable positions were represented as a set of backboneindependent side-chain rotamers derived from the rotamer library of Dunbrack and Cohen (20).…”

“…SPA calculations generated a list of 300 sequences that were subsequently clustered computationally into groups of similar sequences using a nearest neighbor single linkage hierarchical clustering algorithm. Parameters and other details for PDA and SPA calculations are described elsewhere (13,(17)(18)(19) and in unpublished results.…”

“…One set of variants was designed using Protein Design Automation (PDA) (13,17,18) and Sequence Prediction Algorithm (SPA) (19) calculations to optimize the free energy of the ICAM-1·LFA-1 complex. To preserve key contacts at the ICAM-1/LFA-1 interface, Glu-34, Lys-39, Asn-68, and Gln-73 of ICAM-1 were fixed as wild-type in the calculations (8).…”

“…Recent advances in computational protein design algorithms (10)(11)(12) have markedly improved capabilities for generating novel proteins with optimized properties, including enhanced stability (13), altered substrate specificity (10), improved binding affinity (14,15), and optimized pharmacokinetics (16). We have taken multiple structure-based approaches to design ICAM-1 variants with enhanced affinity for α L β 2 .…”

Bioinformatic method for protein thermal stabilization by structural entropy optimization

“…Despite many successes and recent technology development, directed evolution still has some practical limitations and thus can be labor-intensive and expensive. Efforts have also been made to computationally design thermally stable proteins, some requiring the three-dimensional structure of the target to be known (10,11). The ''consensus approach'' is a sequence-based method for protein thermal stabilization that seeks to find commonality within a protein family (12).…”