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

Abstract

Ribonucleotide reductase (RR), an established cancer target is usually inhibited by antimetabolites which display multiple cross-reactive effects. Recently, we discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH or E-3a) of human RR (hRR) binding at the catalytic site (C-site) and inhibiting hRR reversibly. We herein report the synthesis and biochemical characterization of 25 distinct analogs. We designed each analog through docking to the C-site of hRR based on our 2.7Å X-ray crystal structure (PDB ID: 5TUS). Broad tolerance to minor structural variations preserving inhibitory potency is observed. E-3f (82% yield) displayed an in vitro IC50 of 5.3 ± 1.8 µM against hRR, making it the most potent in this series. Kinetic assays reveal that E-3a, E-3c, E-3t and E-3w bind and inhibit hRR through a reversible and competitive mode. Target selectivity towards the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.

Structure-guided design of anti-cancer ribonucleotide reductase inhibitors

“…A hRRM1 dimer with lead compound NSAH bound to the C-site (PDB ID: 5TUS) was used as a model for the docking of the new compounds 35 . It was assumed that the primary site of binding for these compounds would be the C-site, because NSAH and all previously studied derivatives of NSAH have demonstrated a preference for the C-site 35 , 37 . Our studies showed that compounds TP2–7 , 9–10 are longer and therefore extend beyond NSAH, allowing for the possibility of more interactions (Figure 3(A–D) and Supplemental Figure 1(B–F)).…”

“…In silico docking of the compounds was performed using the Glide module of the Schrödinger 2017–3 modelling software suite as previously described 36 , 40–42 . The docking site for the C-site was defined as a 5 Å box centred on the lead compound NSAH ( TP8 ) bound to the hRRM1 (PDB code 5TUS) 37 . Compounds were docked to the C-site using Glide SP.…”

“…Nevertheless, medicinal chemistry and synthetic approaches can be used to improve the potency of NSAH and its target selectivity towards hRR. Indeed, a recent paper from our laboratory reported on a library of 25 NSAH analogues whose modifications were designed to target residues within the C-site of hRR that are important for interactions with natural substrates 37 . These results indicated that those analogues which demonstrated a 2–4 fold improved potency of cell-free hRR over NSAH all showed hydrogen bonding with Ser606, Thr607, and Ser217, residues that are known to hydrogen bond with natural substrates.…”

Still the most interesting enzyme in the world

“…Accordingly, investigators are seeking inhibitors that target RNR more specifically. Two recent papers (13,14) describe exciting results. Ahmad et al (13) and Huff et al (14) described a naphthyl salicyl acyl hydrazone-based compound that inhibits human RNR through binding at the catalytic site.…”

“…Ahmad et al (13) and Huff et al (14) described a naphthyl salicyl acyl hydrazone-based compound that inhibits human RNR through binding at the catalytic site. Huff et al (14) synthesized 25 analogs, each selected for its docking to human R1 protein, based on the crystal structure of the protein determined by the same group. The most effective analog inhibited human RNR activity with a half maximal inhibitory concentration of 5.3 mM.…”