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. Last 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 demonstrated that these are allosteric inhibitors of Ubc9 thioester formation that probably function by rigidifying the protein. This year we collaborated with Dr. Schneekloth's group on the identification of additional Ubc9 inhibitors by small molecule microarray screening. 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. These are the first crystal structures of TDP1 in complex with any small molecule inhibitors. We also utilized Dr. Schneekloth's small molecule microarray to identify additional inhibitors of TDP1, and these are currently being optimized in collaboration with Dr. Terrence Burke's group. 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. Over the years we have determined the structures of a number of protein phosphates. This year we published the structure of the human dual specificity phosphatase 1 (DUSP1) catalytic domain. DUSP1 is the DUSP that is most frequently associated with cancer and is therefore an attractive drug target. 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 previously determined three crystal structures of the catalytically inactive MERS-CoV 3CLpro enzyme. Current effort is focused on identifying inhibitors of this enzyme as well as 3CLpro enzymes from five other coronaviruses that infect humans with the goal of developing a broad spectrum coronavirus inhibitor. Several compounds with IC50 values in the low micro molar range have been identified recently. Our collaborator on this project is Dr. Robert Ulrich at USAMRIID.
| Waugh, David S (2016) Crystal structures of MBP fusion proteins. Protein Sci 25:559-71 |
| 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 |
| 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 |
| 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; 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 |
| 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 |
