Small Ubiquitin-like Modifier (SUMO) modification is a post-translational modification affecting many cellular processes, including nuclear transport, transcriptional regulation, cell cycle progression, and protein stability. Upregulation of SUMO, as well as other components of the SUMOylation pathway, has been observed in human cancers such as lung adenocarcinomas, anaplastic large-cell lymphoma, hepatocellular carcinoma, and ovarian tumors. Since SUMO modification changes the activity of a large number of proteins, SUMOylation pathway is one of the cancer-supportive cellular machineries responsible for many aspects of abnormal cell functions in cancer including increased survival of cancerous cells. We hypothesized that targeting SUMOylation may decrease cancer cells' viability. Recently, our lab used FRET-based high-throughput screening to identify STE as an inhibitor of the SUMOylation pathway.

To elucidate the mechanism of action of STE, we performed SUMO E1 and E2 thioester formation assays and the enzyme kinetic assays. STE inhibits E1~SUMO thioester conjugate formation in a dose dependent manner and this inhibition is specific to the SUMOylation pathway. We developed a quantitative FRET technology to measure the E1 enzyme kinetics and employed this method to measure the inhibition constant of the STE. We found the results of enzyme kinetics assays using the quantitative FRET methods are comparable to those of conventional radioactive assays. The Km of SUMO2 (3.42 ± 0.91 µM) and SUMO3 (2.76 ± 0.75 µM) are about four to five times higher than the Km of SUMO1 (0.75 ± 0.11 µM). In the presence of STE, the Km for the E1 enzyme is unaltered whereas the Vmax is decreased as STE concentration increases which indicates that STE is a non-competitive inhibitor of the E1 enzyme. Based on these experiments, the inhibition constant of the STE was calculated to be 1.90µM. Our results also indicated that STE induces cell death in the both Non-Small Cell Lung Cancer (NSCLC) cell lines NCI-H358 and NCI-H460 and inhibit cell cycle progression in HEK293.

We identified and characterized STE, a small molecule inhibitor of the E1 enzyme of the SUMOylation pathway that is active in the cells. Due to the diverse roles of SUMO, the identification of selective inhibitors of the SUMOylation pathway may lead to pharmacological tools for cancer treatment.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.