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

Proc Natl Acad Sci U S A. 2017 Aug 1;114(31):8241-8246. doi: 10.1073/pnas.1620220114. Epub 2017 Jul 17.

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 IC50 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.

Keywords: cancer chemotherapy; drug discovery; enzyme regulation; ribonucleotide reductase; small molecule.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalytic Domain
  • Cell Cycle / drug effects
  • Crystallography, X-Ray
  • Deoxyadenine Nucleotides / metabolism
  • Drug Screening Assays, Antitumor / methods
  • Humans
  • Hydrazones / chemistry
  • Hydrazones / pharmacology*
  • Naphthalenes / chemistry
  • Naphthalenes / pharmacology*
  • Ribonucleoside Diphosphate Reductase
  • Ribonucleotide Reductases / antagonists & inhibitors*
  • Ribonucleotide Reductases / chemistry
  • Ribonucleotide Reductases / metabolism
  • Salicylates / chemistry
  • Salicylates / pharmacology*
  • Tumor Suppressor Proteins / antagonists & inhibitors
  • Tumor Suppressor Proteins / chemistry
  • Tumor Suppressor Proteins / metabolism

Substances

  • Deoxyadenine Nucleotides
  • Hydrazones
  • Naphthalenes
  • Salicylates
  • Tumor Suppressor Proteins
  • Ribonucleotide Reductases
  • RRM1 protein, human
  • Ribonucleoside Diphosphate Reductase
  • 2'-deoxyadenosine triphosphate

Associated data

  • PDB/5TUS