ABSTRACT
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 30-120â nM) in an inâ 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 10â µM. Evidence for a druggable allosteric pocket in human ATC is provided by both inâ 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.
Subject(s)
Aspartate Carbamoyltransferase , Cell Proliferation , Enzyme Inhibitors , Osteosarcoma , Humans , Cell Proliferation/drug effects , Osteosarcoma/drug therapy , Osteosarcoma/pathology , Osteosarcoma/metabolism , Aspartate Carbamoyltransferase/antagonists & inhibitors , Aspartate Carbamoyltransferase/metabolism , Aspartate Carbamoyltransferase/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemical synthesis , Allosteric Regulation/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Structure-Activity Relationship , Dose-Response Relationship, Drug , Molecular Docking Simulation , Molecular Structure , Drug Screening Assays, Antitumor , Cell Line, Tumor , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , Bone Neoplasms/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolismABSTRACT
The discovery and development of new drugs against malaria remain urgent. Aspartate transcarbamoylase (ATC) has been suggested to be a promising target for antimalarial drug development. Here, we describe a series of small-molecule inhibitors of P. falciparum ATC with low nanomolar binding affinities that selectively bind to a previously unreported allosteric pocket, thereby inhibiting ATC activation. We demonstrate that the buried allosteric pocket is located close to the traditional ATC active site and that reported compounds maintain the active site of PfATC in its low substrate affinity/low activity conformation. These compounds inhibit parasite growth in blood stage cultures at single digit micromolar concentrations, whereas limited effects were seen against human normal lymphocytes. To our knowledge, this series represent the first PfATC-specific allosteric inhibitors.