Insights into the inhibition of ETV6 PNT domain polymerization

ETV6 is a modular transcriptional repressor for which head-to-tail polymerization of its PNT (or SAM) domain facilitates cooperative binding to tandem DNA sites by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to the catalytic domain of one of several receptor protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, auto-phosphorylation, and aberrant stimulation of downstream signaling pathways, thereby leading to a diverse range of cancers. Inhibition of PNT domain polymerization through mutations renders the chimeric proteins non-oncogenic. This indicates that a small molecule inhibitor of polymerization could be a viable therapeutic against many ETV6-linked cancers. Protein-protein interactions are challenging to disrupt with small molecules, and thus I followed a multi-pronged approach for lead compound discovery. In Chapter 2, I describe work to obtain structural, dynamic, and thermodynamic insights into the PNT domain and its self-association properties. To this end, I characterized monomeric and heterodimeric forms of the PNT domain using nuclear magnetic resonance spectroscopy, X-ray crystallography, molecular dynamics simulations, amide hydrogen exchange, and alanine scanning mutagenesis in conjunction with surface plasmon resonance binding studies. Collectively, these studies defined “hot spot” regions critical to the PNT domain self-association interface. In Chapter 3, I discuss efforts undertaken to find inhibitors of ETV6 PNT domain polymerization using two high-throughput cellular approaches – a split luciferase reporter assay and a modified yeast two-hybrid assay – and a computational approach with the Bristol University Docking Engine (BUDE). Over 75 lead compounds from these assays were tested for binding to the PNT domain through NMR spectroscopy. None were found to bind to the PNT domain or inhibit its self-association. However, lessons learned from these screening assays may facilitate future high-throughput screening for or rational design of therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

The antimalarial drug mefloquine enhances TP53 premature termination codon readthrough by aminoglycoside G418.

Nonsense mutations constitute ~10% of TP53 mutations in cancer. They introduce a premature termination codon that gives rise to truncated p53 protein with impaired function. The aminoglycoside G418 can induce TP53 premature termination codon readthrough and thus increase cellular levels of full-length protein. Small molecule phthalimide derivatives that can enhance the readthrough activity of G418 have also been described. To determine whether readthrough enhancers exist among drugs that are already approved for use in humans, we tested seven antimalarial drugs for readthrough of the common R213X TP53 nonsense mutation in HDQ-P1 breast cancer cells. Mefloquine induced no TP53 readthrough activity as a single agent but it strongly potentiated readthrough by G418. The two enantiomers composing pharmaceutical mefloquine potentiated readthrough to similar levels in HDQ-P1 cells and also in SW900, NCI-H1688 and HCC1937 cancer cells with different TP53 nonsense mutations. Exposure to G418 and mefloquine increased p53 phosphorylation at Ser15 and P21 transcript levels following DNA damage, indicating p53 produced via readthrough was functional. Mefloquine does not appear to enhance readthrough via lysosomotropic effects as it did not significantly affect lysosomal pH, the cellular levels of G418 or its distribution in organellar or cytosolic fractions. The availability of a readthrough enhancer that is already approved for use in humans should facilitate study of the therapeutic potential of TP53 readthrough in preclinical cancer models

Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases

The glycoside hydrolase family 4 (GH4) α-galactosidase from <i>Citrobacter freundii</i> (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD⁺ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived β_lg value on <i>V</i>/<i>K</i> for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr²⁺, Y³⁺, and Mn²⁺, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured ^D<i>V</i>/<i>K</i> KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-galactopyranoside are closer to unity than the measured effects on 4-fluorophenyl α-d-galactopyranoside, irrespective of the site of isotopic substitution and of the metal cation activator. These observations are consistent with hydride transfer at C-3 to the on-board NAD⁺, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for <i>V</i>/<i>K</i>