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. 2018 Feb 8;61(3):666-680.
doi: 10.1021/acs.jmedchem.7b00530. Epub 2018 Jan 5.

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

Sarah E Huff  1 , Faiz Ahmad Mohammed  2 , Mu Yang  1 , Prashansa Agrawal  1 , John Pink  3 , Michael E Harris  4 , Chris G Dealwis  2   5 , Rajesh Viswanathan  6   1
Affiliations

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

Sarah E Huff et al. J Med Chem. .

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 2.7 Å 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 toward the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests

Figures

Figure 1
Figure 1
A. Conversion of ribonucleoside diphosphates into deoxyribonucleoside diphosphates by RR and its redox coupled activity with thioredoxin; B. Comparison of lead compound Naphthyl Salicyl Acyl Hydrazone (NSAH or E-3a) and natural substrate GDP bound to the C-site of hRR (PDB ID: 5TUS); C. Overall approach leading to identification of novel hRR inhibitor whose analogs (E-3a-z) are synthesized and biochemically characterized in this report.
Figure 2
Figure 2
Predicted binding interactions for top-ranked NSAH candidates at the C-site of hRR based on the X-ray recently published structure PDB ID: 5TUS. A. E-3a; B. E-3c; C. E-3f; D. E-3s; E. E-3t; F. E-3u.
Figure 3
Figure 3
A. Double-reciprocal plot for E-3a. All data sets converge upon a common y- intercept, supporting a competitive mechanism of inhibition. B. Double-reciprocal plot for E-3c also follows a competitive model. C. Double-reciprocal plot for E-3t, depicting competitive inhibition. D. Double-reciprocal plot for E-3w supports a competitive model of inhibition.
Figure 4
Figure 4
Sigmoidal dose-response curves for triapine and E-3a against hRRM2. The IC50 values of triapine and E-3a are 0.185 and 123 µM, respectively.
Figure 5
Figure 5
A and B. Sensitization of MDA-MB-231 cell line toward NSAH inhibitor under siRNA transfection and RRM1 knockdown conditions. A: Using RRM1 siRNA and B: Using Control-scrambled siRNA. Line displayed in A at the 4.0 pmol level represents the maximal sensitization occurring in the presence of E-3a, operating within an optimized siRNA concentration range.
Scheme 1
Scheme 1
A. Modular synthesis of hRR inhibitor library.; Yields and other structural characterization per each analog is provided in the experimental section. Thermodynamic control of the condensation step leading to E-isomer is observed. B. UV profile for E-3a. λmax at 320 nm is attributed to acyl hydrazone in trans configuration. The varying concentrations for E-3a are 2.0, 4.0, 8.0, 16.0 and 32.0 µM respectively as measured in MeOH as a solvent. Data corrected for solvent and background.
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