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Villanueva, Nicolas L.

Cell and Molecular Biology Program

2012

Ph. D.

Despite years of improvement and dramatic advancess in the field of drug discovery, one class of targets has remained elusive. Proteins that mediate disease through interactions with other macromolecules are commonly considered "undruggable" or at least sufficiently difficult to warrant extreme caution and often an avoidance of them as the target of a drug discovery team. A stumbling block in the effort to discover small molecules that can be worked into drugs against these targets is that there is rarely an obvious starting point from which to construct a ligand. The Pharmaceutical industry is well equipped to target enzymes that interact primarily through small molecules, in part because often the natural substrate can act as an initial scaffold to elaborate toward a drug. In the case of a protein that binds to other macromolecules this small molecule starting point is absent and a different approach must be taken. Additional challenges in the case of a protein-macromolecule mediated interaction are that the binding surface is often undefined and a large surface is required to provide the energy of binding. This problem is absent for the macromolecular binding partner but it represents a significant problem when one is attempting to develop a small molecule therapeutic.
The recently adopted techniques of screening for small molecules that can target sites of interest using very small molecule fragments in conjunction with structure guided design of small molecules toward drugs provide an opening for fmding and optimizing small molecules against macromolecular binding surfaces on target proteins. Fragments are often found to bind to natural ligand binding sites. This innate ability allows the fragment to overcome the hurdle of simply fmding the target site. Additionally, due to their small size fragments have plenty of room to develop toward a drug with reasonable properties. However, fragments are composed of so few groups that there are not many possible interactions they can make with the target site so their affmity tends to be quite low. Binding ofa low affinity ligand is difficult to detect above noise in an assay.
This work aims to develop X-ray crystallographic techniques that will make the discovery of small molecules that act on macromolecular interfaces a more tenable goal. The Rad51 protein is an example of a protein that mediates genetic changes through Ii series of macromolecular interactions. Though Rad51 is an enzyme the active site is not fully formed unless it is in complex with another Rad51 protomer, the crystallographic and biochemical study of a mutation at this interface active site provides insights into the mechanism of this enzyme that could be a drug target. Increasing the sensitivity of a crystallographic binding assay is then pursued through the use of X-ray contrast enhanced molecules. A technique that allows for the discovery of small weakly binding ligands using X-ray contrast enhanced molecules as an atomic beacon to signal binding is developed. Finally, using probe ions, and a site directed mutation to the electrostatic field of a model protein, an empirical test of the range of the electrostatic field is pursued. This experimental data could provide justification for theoretical models of the atomic force field and allow for more robust in silico screening for protein-ligand interactions.