With Dr. John (Jay) Schneekloth (Chemical Biology Laboratory, CCR), we are attempting to develop inhibitors/chemical probes of Ubc9, the lone SUMO-conjugating enzyme in humans. SUMOylation regulates a wide variety of biological processes, and defects in SUMO homeostasis are associated with many diseases including cancer. High-resolution crystal structures of Ubc9 with drug-like ligands are being sought and obtained by fragment screening and other complementary approaches. This year we identified two fragments that bind to the same site that is on the opposite side of the protein from its active site. Subsequent studies have shown that these are allosteric inhibitors of Ubc9 thioester formation that probably function by rigidifying the protein. Human tyrosyl-DNA phosphodiesterase 1 (Tdp-1) is the focus of another project. Tdp1 inhibitors are expected to act synergistically with topoisomerase 1-targeting drugs (camptothecins, indenoisoquinolines), topoisomerase 2 inhibitors (etoposide, doxorubicin), bleomycin, and DNA alkylating agents that are already used for cancer chemotherapy. In collaboration with Dr. Yves Pommier (Developmental Therapeutics Branch, CCR), we are seeking to elucidate the structural basis for inhibition of Tdp-1 by various compounds and, by extension, gain insight into how these inhibitors might be improved by rational design. We recently identified two fragments by crystallographic screening that bind in the active site of Tdp-1 and engage its active site residues. We are currently in the process of optimizing these lead compounds. Protein phosphorylation is a reversible event that is carefully controlled by the opposing activities of kinases and phosphatases. Abnormal changes to this equilibrium often lead to deleterious effects such as deregulated cell growth and cancer. We are collaborating with Dr. Terrence Burke Jr. (Chemical Biology Laboratory, CCR), an expert in the field of protein tyrosine phosphatase (PTPase) inhibition, to develop inhibitors of PTPase epsilon, a membrane-bound enzyme that is up-regulated in human breast cancer cells and which is also a negative regulator of insulin receptor signaling. To facilitate this goal, we have recently determined high-resolution crystal structures of both the membrane-proximal catalytic domain and the membrane-distal inactive PTPase domain. Novel tri- and tetra-dentate inhibitors are being synthesized by oxime-based chemistry and screened for inhibition of PTPase epsilon. Crystal structures of the dual specificity phosphatases DUSP7 and DUSP22 have also been determined. The Middle East Respiratory Syndrome Coronavirus [MERS-CoV] is a highly pathogenic virus that causes severe respiratory illness accompanied by multi-organ dysfunction, resulting in a case fatality rate of approximately 40%. Like other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into a long polyprotein that is cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3CLpro). Since 3CLpro is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3CLpro inhibitors, we determined three crystal structures of the catalytically inactive MERS-CoV 3CLpro enzyme. Current effort is focused on identifying inhibitors of this enzyme by fragment screening. Extramural collaborators include investigators at the United States Army Medical Institute of Infectious Diseases (Drs. Robert Ulrich and Kamal Saikh), with whom we are studying the role of the adaptor molecule MyD88 in the innate immune response and structure/function relationships in dual-specificity phosphatases.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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Hewitt, William M; Lountos, George T; Zlotkowski, Katherine et al. (2016) Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9. Angew Chem Int Ed Engl 55:5703-7
Waugh, David S (2016) Crystal structures of MBP fusion proteins. Protein Sci 25:559-71
Olson, Mark A; Lee, Michael S; Kissner, Teri L et al. (2015) Discovery of small molecule inhibitors of MyD88-dependent signaling pathways using a computational screen. Sci Rep 5:14246
Lountos, George T; Cherry, Scott; Tropea, Joseph E et al. (2015) Structural analysis of human dual-specificity phosphatase 22 complexed with a phosphotyrosine-like substrate. Acta Crystallogr F Struct Biol Commun 71:199-205
Zhao, Bryan M; Keasey, Sarah L; Tropea, Joseph E et al. (2015) Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases. PLoS One 10:e0134984
Needle, Danielle; Lountos, George T; Waugh, David S (2015) Structures of the Middle East respiratory syndrome coronavirus 3C-like protease reveal insights into substrate specificity. Acta Crystallogr D Biol Crystallogr 71:1102-11
Lountos, George T; Austin, Brian P; Tropea, Joseph E et al. (2015) Structure of human dual-specificity phosphatase 7, a potential cancer drug target. Acta Crystallogr F Struct Biol Commun 71:650-6