Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.

Adviser: Jonathan A. Ellman.

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This dissertation describes the work I have completed over the course of my graduate studies in the Ellman lab on two separate project areas. Chapter 1 serves as an introduction to the first project area, the enzymes in the protein arginine deiminase family. Chapter 2 describes the discovery and optimization of the first examples of small molecule inhibitors for protein arginine deiminase 3 using a substrate-based fragment screening method. Chapter 3 describes further optimization and subsequent demonstration of in vivo activity for these inhibitors. Following completion of this project, my work focused on the discovery of inhibitors of MAP Kinase Phosphatase 5. Chapter 4 describes a high-throughput screen leading to the identification of allosteric inhibitors of this enzyme.

Chapter 1 introduces protein arginine deiminases (PADs), which are a family of enzymes that catalyze the post-translational modification of arginine residues. The mechanism of PAD catalysis is described. PAD structure, cellular substrates, and physiological roles are then outlined, followed by a discussion of PAD small molecule inhibitors described in the literature. Finally, PAD3, the isozyme within the PAD family that is the focus of this research, is discussed in detail as a promising therapeutic target.

Chapter 2 describes the development of an approach for the reliable discovery of low molecular weight, nonpeptidic fragment substrates of the PADs that then can be optimized and converted to mechanism based irreversible PAD inhibitors. The approach is demonstrated by the development of potent and selective inhibitors of PAD3, a PAD subtype implicated in the neurodegenerative response to spinal cord injury. Multiple structurally distinct inhibitors were identified with the most potent inhibitors having >10,000 min-1 M-1 k inact/KI values and ≥10-fold selectivity for PAD3 over PADs 1, 2, and 4.

Chapter 3 describes the design, synthesis and evaluation of conformationally constrained versions of a potent and selective PAD3 inhibitor that we had previously reported resulting in the identification of more potent PAD3 inhibitors. PAD3 has been characterized as a modulator of cell growth via apoptosis inducing factor. The cell activity of representative inhibitors in this series was therefore demonstrated for the first time by rescue of thapsigargin-induced cell death in PAD3-expressing HEK293T cells.

Chapter 4 introduces Duchenne Muscular Dystrophy (DMD) as a progressive neuromuscular disorder causing debilitating muscle weakness. DMD is caused by mutations in the DMD gene, which encodes for the dystrophin protein, an important component of muscle fiber cells. No cure for DMD currently exists. MAP Kinase Phosphatase 5 (MKP5) has recently been identified as a target for the treatment of DMD. This chapter furthermore describes a small molecule screen that led to the identification of a lead compound that inhibits MKP5. A crystal structure of the lead compound in complex with MKP5 is reported. Furthermore, analogs of the hit compound were synthesized to enable the discovery of soluble and potent inhibitors of MKP5.