Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Medicine

Major Professor

Yu Chen, Ph.D.

Committee Member

Robert J. Deschenes, Ph.D.

Committee Member

Vladimir Uversky, Ph.D.

Committee Member

Wayne C. Guida, Ph.D.


Virtual Screening, Molecular Docking, X-ray Crystallography, Structural Biology


For structure-based drug discovery, structural information of a target protein is necessary. NMR, or X-ray crystallography can provide necessary information on active site configuration that can lead a successful virtual screening campaign into identifying binders that may then be optimized into potent inhibitors. However, many challenges exist in the structure-based drug discovery cycle. For instance, structure determination of a protein of interest can many times be a daunting task. In addition, complex structure determination, which can allow essential characterization of protein-ligand interactions, is also challenging and many times impossible. Virtual screening heavily relies on such structural information, but hit-to-lead optimization schemes do as well. Furthermore, inherent protein characteristics such as conformational flexibility only add to the complexities in using structural information to identifying and optimizing inhibitors. In the scope of the work presented here, a structure-based drug discovery approach against three different protein targets is described. Each is presented with it's own set of challenges, but each has successfully led to the identification of new ligands.

The drug discovery project against CXCL12 will first be described. CXCL12 is a small chemokine (~10KDa) that binds to the CXCR4 receptor promoting chemotaxis of lymphocytes but also metastasis of cancer cells. This interaction is further supported by sulfated tyrosines on CXCR4 that bind specific sites on the CXCL12 surface. The CXCL12-CXCR4 signaling axis has been a major focus of drug discovery, but efforts are mainly focused on CXCR4, since CXCL12 is a small protein lacking surface characteristics that are thought to be druggable. Yet, through a combination of rigid, flexible, and ensemble docking in virtual screening studies, we have successfully identified compounds that bind each of the three sulfotyrosine recognition sites on CXCL12, which normally bind the sulfated tyrosines on CXCR4 (sY7, sY12, and sY21). Furthermore, we have led a hit-to-lead approach in optimizing compounds against the sY21-binding site, aided by trivial information gained through crystallographic complex structure determination of CXCL12 bound by such a compound. We aim to eventually link compounds against different sites together and greatly improve potency.

Next, the drug discovery project against P. aeruginosa LpxA will be described. In Gram-negative bacteria, the first step of lipid A biosynthesis is catalyzed by UDP-N-acetylglucosamine acyltrasferase (LpxA) through the transfer of a R-3-hydroxyacyl chain from the acyl carrier protein (ACP) to the 3'-hydroxyl group of UDP-GlcNAc. Acyl chain length selectivity varies between species of bacteria, but is highly specific and conserved within certain species. In E. coli and L. interrogans for example, LpxA is highly selective for longer R-3-hydroxyacil chains (C14 and C12 respectively), while in P. aeruginosa the enzyme is highly selective for R-3-hydroxydecanoyl, a 10-hydrocarbon long acyl chain. Three P. aeruginosa LpxA crystal structures will be described here for the first time; the apo form, the complex with its substrate UDP-GlcNAc, and the complex with its product UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc. A comparison between the APO form and complexes identifies key residues that position UDP-GlcNAc appropriately for catalysis, and supports the role of His121 in generating the nucleophile by interacting with the UDP-GlcNAc 3'-hydroxyl group. Furthermore, the product-complex structure supports the role of Met169 as the "hydrocarbon ruler", providing structural information on how P. aeruginosa LpxA is granted its exceptional selectivity for the 10-hydrocarbon long acyl chain. Structural information of the active site was subsequently used in designing virtual screening experiments that led to the identification of two ligands, confirmed by X-ray crystallography screening to bind to the active site. We aim to continue application of X-ray crystallography into screening compound binding, and to also use a hit-to-lead approach in compound optimization.

Finally, the drug discovery project against the Tiam1 PDZ domain will be described. Tiam1 (T-cell lymphoma invasion and metastasis gene 1) is a GEF (guanine exchange factor) protein that activates Rac1 and initiates tumor formation. Tiam1 is regulated through its PDZ domain, which binds to syndecan1. We have successfully applied a virtual screening strategy to an existing crystallographic structure of the Tiam1 PDZ domain complexed to a syndecan1 peptide and identified four ligands that bind to the PDZ domain with low affinities. These compounds provide a starting point for future hit-to-lead optimization strategies.