Date of Award

12-4-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Bioinformatics

First Advisor

Kottayil Varughese

Abstract

Computer-aided rational drug design is one of the most promising approaches for the development of effective drug therapies. This dissertation describes applications of rational structure-based techniques in two cases. In the first study, we enhanced the potency of a therapeutic antibody against methamphetamine. In the second study we identified potentially therapeutic molecules to treat atherosclerosis. Methamphetamine abuse is a serious health hazard and a single chain variable fragment (scFv) of a therapeutic anti-METH antibody has been developed at this University. Anti-METH immunotherapy blocks or reduces the rate of METH entry into the brain. In this research we introduced rationally designed point mutations in the binding pocket to enhance the affinity towards METH and its active metabolite amphetamine (AMP). One mutant, scFv-S93T, showed a 3.1 fold enhancement in affinity for METH and a 26 fold enhancement for AMP. Two other mutants scFv-I37M and scFv-Y34M showed enhancements of 94, and 8 fold for AMP, respectively. Structural analysis of the scFv-S93T:METH complex revealed that the mutation caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment in the binding pocket. We also applied computer-aided rational drug design techniques to identify anti-atherosclerotic drugs using the lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) as a target. Atherosclerotic related diseases are a major cause of death in the United States. LOX-1 mediates the internalization of ox-LDL in endothelial cells. This key event causes endothelial dysfunction and plaque formation and leads to the development of atherosclerosis. In this research, we used the high-resolution crystal structure of LOX-1 to perform rational virtual screening and docking studies to identify novel inhibitors of LOX-1 with the goal to inhibit LOX-1 and ox-LDL interaction. Leads were tested for in vitro binding affinity for LOX-1, using thermal shift assays and the top two leads increased the melting temperature by 9(±2)°C and 4(±1)°C respectively. In addition, the cell-based assays (Dr. Mehta lab) demonstrated that the lead compounds inhibit LOX-1 and prevent/reduce the internalization of ox-LDL in endothelial cells.

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