Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule

Abstract

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 μM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC 50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.ribonucleotide reductase | cancer chemotherapy | small molecule | drug discovery | enzyme regulation

Structure-Guided Synthesis and Mechanistic Studies Reveal Sweetspots on Naphthyl Salicyl Hydrazone Scaffold as Non-Nucleosidic Competitive, Reversible Inhibitors of Human Ribonucleotide Reductase

“…Detailed kinetics assays reveal that these inhibitors bind and inactivate hRR through a reversible and competitive mode, consistent with our recently reported finding for the lead inhibitor E - 3a . 10 Remarkably, this class of compounds are the first non-nucleosidic inhibitors of hRR displaying a competitive, reversible mode of inhibition, consistent with C-site binding.…”

“…We noted the key residues involved in NDP binding in the C-site of hRR and catalysis, such as: Glu 431, Cys 429, Cys 444, Cys 218 and Asn 427. Upon comparing the close hydrogen bonding contacts between parent inhibitor ( E - 3a ) at the C-site of hRR in our recent X-ray structure (PDB ID: 5TUS) 10 , Ser 217 and Cys 218 made relevant contacts through hydrogen bonds with E - 3a . Cys 429 offered weaker Van der Waals contact with inhibitor E - 3a , however it did not contribute strongly to stabilize binding to the inhibitor.…”

“…10 Approximately one hundred distinct analogs with unique substitution patterns surrounding the central hydrazone core were docked to the C-site, where the grid was defined as a 5 Å cube centered around the lead hydrazone ligand. Evaluation of the docking poses for interactions with residues previously established to interact with nucleoside substrates, such as Ser 202, Ser 606, Gly 246, Cys 218, Cys 429 and Ser 217, helped define a subset of twenty five analogs most likely to bind at the C-site and inhibit the enzyme.…”

“…10 E - 3a demonstrated significant levels of selective cytotoxicity against multiple cancer cell lines while displaying little cytotoxicity against normal mobilized peripheral blood progenitor cells. 10 This result presented us with a lead compound possessing favorable properties worthy of further exploration.…”

“…10 E - 3a demonstrated significant levels of selective cytotoxicity against multiple cancer cell lines while displaying little cytotoxicity against normal mobilized peripheral blood progenitor cells. 10 This result presented us with a lead compound possessing favorable properties worthy of further exploration. In the present study we use the previously determined 2.7 Å crystal structure of this lead inhibitor ( E - 3a ) in complex with hRRM1 (PDB ID: 5TUS) 10 as a template for structure-based ligand optimization.…”