We have taken a multipronged approach to the identification of novel sumoylation inhibitors. These advances are divided into two areas, based on screening and design-based approaches: 1. Development of a high throughput screen to identify sumoylation inhibitors. A major goal of this project is to carry out a high throughput screen to identify novel small molecule sumoylation inhibitors. My laboratory has developed a completely novel biochemical assay to monitor a reconstituted sumoylation cascade using recombinant proteins. In the past year we have optimized and miniaturized the assay, and completed two pilot screens in collaboration with NCI's Molecular Targets Laboratory (MTL). An initial paper was published earlier in the year, describing a novel mechanism of inhibition of the SUMO pathway as a result of these pilot screens. More specifically, we have identified the first example of a molecule that prevents discharge of SUMO from the UBC9-SUMO thioester. This molecule is active in several biochemical and cell-based assays. We are currently near completion of a high throughput screen, having analyzed 25,000 pure compounds and 100,000 natural product extracts for activity against the SUMO pathway. Once completed, this screen will provide the lead molecules for further medicinal chemistry and/or total synthesis efforts which will be carried out in the lab. After lead compounds are identified, we will pursue their synthesis and characterize their biochemical mechanism of action. Chemistry efforts will focus on the optimization of physical properties such as potency, cell permeability, and solubility, and will be studied using a variety of techniques such as X-Ray crystallographic (In collaboration with David Waugh) and other biophysical means. My laboratory will study enzyme inhibitor kinteics, while several collaborators have already agreed to use identified inhibitors in other, more advanced genetic and mouse model systems to evaluate the anticancer potential of sumoylation inhibitors. 2. Structure based design of novel inhibitors. In collaboration with David Waugh, we are pursuing the structure based design of novel inhibitors of sumoylation enzymes. This is a fragment based drug design approach, whereby we use X-Ray crystallographic analysis of the enzymes to identify fragments capable of interacting with the protein of interest in a defined way. These fragments may then be optimized in to more druglike inhibitors using a medicinal chemistry approach, coupled with biophysical and biochemical analysis. In addition, we are pursuing peptide- and peptidomimetic molecules that bind to sumoylation enzymes in an effort to identify drugable molecular interactions on several enzymes. Most recently, we have identified two fragments that bind to UBC9. We have also developed a number of enzymatic and biophysical (binding) assays to help quantify and improve the activity/affinity of these fragments toward UBC9.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Zlotkowski, Katherine; Hewitt, William M; Sinniah, Ranu S et al. (2017) A Small-Molecule Microarray Approach for the Identification of E2 Enzyme Inhibitors in Ubiquitin-Like Conjugation Pathways. SLAS Discov 22:760-766
Schneekloth Jr, John S (2017) Drug discovery: Controlling protein SUMOylation. Nat Chem Biol 13:1141-1142
Zlotkowski, Katherine; Hewitt, William M; Yan, Pengcheng et al. (2017) Macrophilone A: Structure Elucidation, Total Synthesis, and Functional Evaluation of a Biologically Active Iminoquinone from the Marine Hydroid Macrorhynchia philippina. Org Lett 19:1726-1729
Connelly, Colleen M; Abulwerdi, Fardokht A; Schneekloth Jr, John S (2017) Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays. Methods Mol Biol 1518:157-175
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
Leyva, Melissa J; Kim, Yeong Sang; Peach, Megan L et al. (2015) Synthetic derivatives of the SUMO consensus sequence provide a basis for improved substrate recognition. Bioorg Med Chem Lett 25:2146-51
Suzawa, Miyuki; Miranda, Diego A; Ramos, Karmela A et al. (2015) A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver. Elife 4:
Kim, Yeong Sang; Keyser, Samantha G L; Schneekloth Jr, John S (2014) Synthesis of 2',3',4'-trihydroxyflavone (2-D08), an inhibitor of protein sumoylation. Bioorg Med Chem Lett 24:1094-7
Kim, Yeong Sang; Nagy, Katelyn; Keyser, Samantha et al. (2013) An electrophoretic mobility shift assay identifies a mechanistically unique inhibitor of protein sumoylation. Chem Biol 20:604-13
Kim, Jimin; Schneekloth Jr, John S; Sorensen, Erik J (2012) A chemical synthesis of 11-methoxy mitragynine pseudoindoxyl featuring the interrupted Ugi reaction. Chem Sci 3:2849-2852

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