Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators

Abstract

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.

Figure 1

hRRM1 drug binding sites and fluorescence quenching identifies RR inhibitors. (A) Structure of the hRRM1 dimer with drug-target sites mapped. The M-site is the binding site for the new class of modulators that is the subject of this study. The A-site controls activity. The S-site controls specificity. The C-site is the catalytic site. The P-site binds the smaller R2 subunit derived peptide. (B and C) Tryptophan fluorescence quenching of hRRM1 in the presence of compounds 4 and 10, respectively. (D and E) No tryptophan fluorescence quenching of hRRM1 by compounds 100207 and 184612.

Figure 2

Compound 4 interactions with hRRM1. (A) Model of the hRRM1 hexamer based on the S. cerevisiae RR1 hexamer structure. Ribbon diagram of the hRRM1 hexamer packing arrangement. hRRM1 monomers are green and magenta. All of the four helix ATP-binding cones are red. The 16 N-terminal residues at the hexamer interface are in cyan. Effectors (TTP) bound at the S-site are drawn in brick red spheres, and compound 4 at the hexamer interface is drawn in blue spheres. (B) The |F₀| − |F_c| electron density for the phthalimide compound (blue) in complex with hRRM1 orthorhombic crystals. Density contoured at 3σ defines the phthalimide binding to hRRM1. (C) 2|F₀| − |F_c| electron density (blue) of the phthalimide compound contoured at 1σ after refinement in the phthalimide-hRRM1 orthorhombic complex. (D) Illustration of the A-site and M-site binding by dATP and compound 4, respectively, Compound 4 is shown in magenta, and dATP is in yellow. (E) Lig plot analysis of compound 4 interactions with hRRM1. The phthalimide compound is shown in purple, carbon atoms in phthalimide are shown in black, oxygen atoms are in red, and nitrogen atoms are in blue. An amino acid residue from hRRM1 interacting with the phthalimide is shown in red.

Figure 3

Growth inhibition of MDA-MB-231 and HCT-116 cancer cells in a moderate throughput screen of candidate RR inhibitors. Cells were treated with 3 doses of candidate drugs (1 μM, 10 μM, and 50 μM) for 3 days in a standard growth inhibition assay in 96 well plates. Growth inhibition in duplicate wells of each drug/dose/cell line was assessed by measuring relative DNA content per well compared with that of untreated cells. Drug effect (1-relative growth) is plotted for the 10 μM and 1 μM dose groups (50 μM groups were not included for clarity).

Figure 4

Growth inhibition of cancer cell lines by compound 1. Cells were seeded into 96 well plates (2000 cells/well) and the following day incubated with the indicated concentrations of gemcitabine alone or in combination with compound 1 for 24 h. At this time, drugs were removed and replaced with control media in the gemcitabine alone groups, or compound 1 at the indicated concentrations in the compound 1 alone or combination groups for an additional 48 h. Relative growth was assessed by measuring DNA content in each well. Each drug concentration was assayed utilizing five replicates for each cell line. Results are representative of at least 2 experiments.

Figure 5

Effect of phthalimide binding on oligomerization in hRRM1 using gel filtration chromatography. (A) Chromatogram of hRRM1 with 1 mM concentration of compound 4 is shown in red, where the native hRRM1 in the absence of compound 4 is shown in black. (B) Standard curve for the determination of molecular masses (Mr) of RR. K_av = (V_e − V₀)/(V_t − V₀), where V_e = elution volume, V₀ = void volume, and V_t = total volume. (C) hRRM1 hexamerization in the presence of dATP; at 50 μMdATP, the hexamers species are predominant with a small amount of dimers. The hexamer to dimer ratio based on integration of the peaks is approximately 3-fold. (D) The chromatogram of hRRM1 in the presence of 1 mM compound 4 and 50 μM dATP. The hexamer to dimer ratio based on integration of the peaks is approximately 7-fold.