Wednesday, April 2, 2025
4/2/2025
Identifying phosphoribosylformylglycinamidine synthase inhibitors as a new class of purine antimetabolites - Upregulation of the purine biosynthetic protein phosphoribosylformylglycinamidine synthase (PFAS) promotes liver cancer cell proliferation and is prognostic for lower liver cancer survival rates. There is a fundamental gap in knowledge about how PFAS contributes to the progression of liver cancer. Exacerbating this challenge is a lack of molecular tools for the study of the function of PFAS. This underlines the critical need for the development of specific inhibitors of PFAS activity, both as probes of function and to form the basis for drug development. The long-term goal of our research program is to develop new anti-cancer therapies that regulate metabolic enzymes to extend life and improve patient outcomes. The overall objective of the proposed studies is to identify and validate specific PFAS inhibitors. Our central hypothesis is that pharmacological knockdown of PFAS activity will slow tumor growth in a subset of cancers and lead to improved clinical outcomes. This hypothesis has been formulated on the basis of our own preliminary data that shows PFAS knockdown slows liver cancer cell proliferation and is also supported by substantial literature associating PFAS upregulation and/or overexpression with increased tumor growth and poorer overall prognoses. The rationale for this work is that a specific inhibitor of PFAS is an essential resource needed to address critical gaps in knowledge, rigorously validate this protein as a therapeutic target in cellular, tissue, and animal models, and to serve as the foundation for the development of a novel class of chemotherapeutic. Guided by strong preliminary studies, we will accomplish our overall objective by pursuing two specific aims: 1) To identify small molecule inhibitors of PFAS enzyme activity, and 2) validate the activity and assess the selectivity, mechanism of action (MoA), cytotoxicity, and initial structure- activity relationship (SAR) of the hit compounds. To facilitate this work, we have developed, miniaturized and piloted a new fluorescent assay for PFAS activity and solved a cryo-EM structure of the human PFAS protein. We will support our hit validation with several secondary enzyme- and cell-based assays which, along with extensive cheminformatics and structural studies, will be used to establish potency, selectivity, MoA and initial SAR. The proposed work is innovative, in our opinion, because it will generate the first-of-its-kind inhibitors of a central purine metabolic protein that is strongly correlated with liver cancer survival. Additional innovative aspects include a novel assay, developed for these studies, and EM structures that will be invaluable assets for structure- function studies and in support of drug development. The proposed studies are significant because they are expected to produce validated inhibitors for use in elucidating the role that PFAS, and nucleotide metabolism in general, plays in support and promotion of liver cancer progression. Ultimately, this has the potential to transform liver cancer treatment and improve patient survival by forming the basis for the development of a new class of therapeutic that reduces the activity of PFAS.
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