This proposal utilizes time-resolved and multi-temperature crystallography of polyamine biosynthesis enzymes: ornithine decarboxylase (ODC), spermidine synthase (SPDS), and spermine synthase (SPS) to improve the structural knowledgebase of these enzymes for dynamic structure-based inhibitor design. These enzymes are upregulated in aggressive hepatocellular carcinomas and in childhood neuroblastomas, and ODC provides feedback to the oncogene myc. However, only ODC has an FDA approved inhibitor, and high-throughput inhibitor screening of these enzymes has proven challenging due to the lack of development of applicable assays. Time-resolved crystallography, utilizing the recently developed Mill...

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This proposal utilizes time-resolved and multi-temperature crystallography of polyamine biosynthesis enzymes: ornithine decarboxylase (ODC), spermidine synthase (SPDS), and spermine synthase (SPS) to improve the structural knowledgebase of these enzymes for dynamic structure-based inhibitor design. These enzymes are upregulated in aggressive hepatocellular carcinomas and in childhood neuroblastomas, and ODC provides feedback to the oncogene myc. However, only ODC has an FDA approved inhibitor, and high-throughput inhibitor screening of these enzymes has proven challenging due to the lack of development of applicable assays. Time-resolved crystallography, utilizing the recently developed Millisecond Mix-and-Quench Crystallography (MMQX) technique, will enable the observation of the intermediate structural enzyme states that occur during the catalytic reaction, which will improve our understanding of required motions for enzymatic activity. In parallel with the time-resolved crystallography experiments, we will also perform multi-temperature crystallography experiments to observe the conformational ensembles of the resting states of ODC, SPDS, and SPS to identify allosteric motions and ground state fluctuations with a variety of cofactors, substrates, and products bound. The newly gained structural information from these experiments will be used to inform computationally aided drug development. Inhibitor candidates that demonstrate promise in silico will then be synthesized in collaboration with the CPRIT Synthesis and Drug Lead Discovery Laboratory at Baylor University. In vitro inhibition assays to confirm activity of candidate compounds will be conducted in collaboration with Dr. Margaret Phillips of UT Southwestern, who has experience with related polyamine biosynthesis enzymes in parasitic pathogens. Utilizing these synergistic experimental and computational tools will enable the development of a new class of enzyme inhibitors that target aggressive cancers.

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