Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120 nM) in an in vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10 μM. Evidence for a druggable allosteric pocket in human ATC is provided by both in vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.

CATRIN Department of Innovative Chemistry Palack University 779 00 Olomouc Holice Czech Republic

Department of Biomedicine Aarhus University Ole Worms Alle' 4 8000 Aarhus C Denmark

Department of Pathology and Medical Biology University of Groningen University Medical Center Groningen 9700 RB Groningen The Netherlands

XB20 Drug Design Groningen Research Institute of Pharmacy University of Groningen A Deusinglaan 1 Groningen 9700AV The Netherlands

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Exploring Aspartate Transcarbamoylase: A Promising Broad-Spectrum Target for Drug Development