Synthesis and Characterization of Triazine-Based Chemical Probes
The 1,3,5-triazine is a privileged scaffold in that it is planar and has three-fold symmetry which allows for controlled modification around the ring structure with various substituents. In this thesis, we report on two modular inhibitor libraries that center around a 1,3,5-triazine core scaffolding system, which have been shown to target protein disulfide isomerase A1 (PDIA1), glutaredoxin-3 (GLRX3), and 6-phosphofructo-1-kinase (PFKP). Protein disulfide isomerase A1 (PDIA1) is a thiol-disulfide oxidoreductase localized in the lumen of the endoplasmic reticulum (ER), and is an important folding catalyst and chaperone for proteins in the secretory pathway. PDIA1 contains two active-site domains (a and a’), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. Here, we synthesize a targeted library o second-generation triazine-based inhibitors to optimize the potency and selectivity of our lead compound, RB-11-ca. Characterization of this targeted library afforded an optimized PDIA1 inhibitor, KSC-34, which covalently modifies C53 in the a site of PDIA1 and demonstrates time-dependent inhibition of the reductase activity of PDIA1 in vitro with a kinact/KI = 9.66 x 103 M-1s-1. Interestingly, KSC-34 treatment demonstrated that a-site inhibition led to decreased secretion of amyloidogenic antibody light chain, thus illustrating that site-selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1. In 2014, our lab first reported on RB7, a dichlorotriazine-based electrophilic small molecule which displayed extremely high reactivity and selectivity toward lysine residues in the proteome. Herein, we further on this study by investigating the unique reactivity of RB7 through the synthesis of a second-generation small molecule electrophile library and investigating proteome-wide reactivity in vitro and in situ. This library afforded KSC-46, an RB-7 analogue with p-chlorothiophenol tuning element, which provided optimal proteome reactivity to use as a scaffold for the generation of a targeted library. To take advantage of the tuned reactivity of KSC-46, a second-generation targeted library was generated to target react residues in the proteome. This library yielded two molecules, KSC-56 and KSC-65, which were identified to target glutaredoxin-3 (GLRX3) and 6-phosphofructo-1-kinase (PFKP), respectively. GLRX3 is a cytosolic, monothiol iron-sulfur cluster chaperon protein which relies on two nucleophilic cysteine residues to bind and transfer iron clusters. PFKP is known to catalyze the first irreversible step in glycolysis and regulates the flux of glucose metabolism in the cell, which makes PFKP an attract therapeutic target. KSC-56 was further characterized to bind to Cys261 in the C-terminal glutaredoxin domain of GLRX3.