Probing the rules of molecular recognition through the de novo design of proteins that bind small-molecule drugs.

Project Summary/Abstract: Designing ligand-binding proteins from scratch is the ultimate test of the principles of molecular recognition by proteins. Emerging technologies recently developed in the DeGrado lab, have enabled us, for the first time, to design small molecules in enclosed cavities in fully synthetic, designed proteins, in a single computational step.

Here, we propose to enhance our newfound understanding of protein-small-molecule interactions through the de novo design of a therapeutic-binding protein with high thermostability, binding affinity and specificity, as well as controlled release capabilities. As a proof-of-concept, we will design a protein carrier that tightly and

specifically binds doxorubicin, a prototypical member of the anthracycline class of topoisomerase II inhibitors. This intrinsically fluorescent and highly complex molecule has multiple functional groups to target, closely related structural derivatives, and several commercially available conjugates and carriers, making it an ideal proof of

concept for both the main fundamental design aspect of this project, and the potential drug delivery application. Protein carrier design will be done by testing and extending our newly developed COMBS (Cooperative Motifs for Binding Sites) algorithm, in conjunction with parametric protein design which allows for the precise design of

highly stable helical bundles. We will further enhance our design capabilities by utilizing histidine residues to facilitate drug binding at physiological pH, and enable controlled release in the acidic tumor microenvironment, owing to the pKa of the imidazole side chain. The best computationally scored designs will be bacterially

synthesized to enable rapid screening of their folding and binding capabilities. X-ray crystallography will be carried out to determine structure-function relations and ascertain agreement of the resulting structure with our

designs. These results will be utilized for the iterative design of the carriers. The biological activity of the carriers will be evaluated via cell viability, proliferation, and wound healing assays as well as confocal microscopy. These will inform on future designs of the carriers, and allow us to fine-tune the amount of histidine residues utilized in

doxorubicin binding. Importantly, the carriers will be kept small, to allow for later chemical synthesis to include all D-amino acids, to avoid early proteolysis and associated immunogenicity (as all-D configured peptides are not displayed by major histocompatibility complexes). This function-directed approach will allow us to obtain

atomic-level control of protein-small-molecule interactions, and while this proposal is fundamental in nature, these design principals can ultimately contribute to the development of a new class of carriers. The utilization of this approach for the design of drug carriers is highly compatible with my scientific background, and represents

the first of its many applications. The proposed research offers excellent training opportunities which will be supported by the cooperative ethos of the DeGrado group, combined with our collaboration with Prof. Chen and Prof. Kortemme, and the institutional resources and unique multi-disciplinary environment at UCSF.