Correction BIOCHEMISTRY Correction for “Potent competitive inhibition of human ribo- nucleotide reductase by a nonnucleoside small molecule,” by Md. Faiz Ahmad, Intekhab Alam, Sarah E. Huff, John Pink, Sheryl A. Flanagan, Donna Shewach, Tessianna A. Misko, Nancy L. Oleinick, William E. Harte, Rajesh Viswanathan, Michael E. Harris, and Chris Godfrey Dealwis, which was first published July 17, 2017; 10.1073/pnas.1620220114 (Proc. Natl. Acad. Sci. U.S.A. 114, 8241–8246). The authors note, on page 8243, right column, first paragraph, line 9, “The C7 atom of the hydrazone backbone is an isosteric center that adopts the transisomer configuration (Fig. 2C)” should instead read, “The C7 atom of the hydrazone backbone is an isosteric center that adopts the Z-isomer configuration (Fig. 2C).” Published under the PNAS license. First published November 9, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2021828117 30858 | PNAS | December 1, 2020 | vol. 117 | no. 48 www.pnas.org Downloaded by guest on September 27, 2021 Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule Md. Faiz Ahmada,1, Intekhab Alama,1, Sarah E. Huffb,1, John Pinkc, Sheryl A. Flanagand, Donna Shewachd, Tessianna A. Miskoa, Nancy L. Oleinickc,e, William E. Hartea, Rajesh Viswanathanb, Michael E. Harrisf, and Chris Godfrey Dealwisa,g,2 aDepartment of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106; bDepartment of Chemistry, Case Western Reserve University, Cleveland, OH 44106; cCase Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106; dDepartment of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109; eDepartment of Radiation Oncology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106; fDepartment of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106; and gCenter for Proteomics and the Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106 Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved June 21, 2017 (received for review December 8, 2016) Human ribonucleotide reductase (hRR) is crucial for DNA replication governs substrate specificity, catalysis, and inhibition of this crit- and maintenance of a balanced dNTP pool, and is an established ical enzyme (8, 9, 11–13). cancer target. Nucleoside analogs such as gemcitabine diphosphate Human RR (hRR holocomplex) is an important target for and clofarabine nucleotides target the large subunit (hRRM1) of hRR. cancer chemotherapy. The nucleotide analogs of chemothera- These drugs have a poor therapeutic index due to toxicity caused peutics such as fludarabine, clofarabine, and cladribine target all by additional effects, including DNA chain termination. The discovery three sites of hRRM1 for inhibition (11, 14, 15) (Fig. 1A). In of nonnucleoside, reversible, small-molecule inhibitors with greater contrast, hydroxyurea (HU) blocks catalysis by targeting the di- specificity against hRRM1 is a key step in the development of more iron cluster of RR2 (16, 17). Gemcitabine diphosphate, clofar- effective treatments for cancer. Here, we report the identification abine, and cladribine nucleotides inhibit hRRM1 by stabilizing a and characterization of a unique nonnucleoside small-molecule hRR form of the α6 complex (10, 11, 13, 15). Such nucleotide-analog inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual inhibitors of hRR, especially gemcitabine, are effective antican- screening, binding affinity, inhibition, and cell toxicity assays. NSAH cer agents and can sensitize cancer cells to ionizing radiation and BIOCHEMISTRY binds to hRRM1 with an apparent dissociation constant of 37 μM, to DNA-damaging drugs (18). Indeed, gemcitabine is a mainstay and steady-state kinetics reveal a competitive mode of inhibition. of the cancer chemotherapy toolbox and is combined with radi- A 2.66-Å resolution crystal structure of NSAH in complex with ation and/or another chemotherapeutic agent, such as cisplatin, hRRM1 demonstrates that NSAH functions by binding at the catalytic in clinical protocols for multiple types of cancer (19). site (C-site) where it makes both common and unique contacts with In addition to its direct anticancer effects, hRR inhibition also the enzyme compared with NDP substrates. Importantly, the IC50 for aids the ability of nucleotide analogs to be incorporated into DNA. NSAH is within twofold of gemcitabine for growth inhibition of Inhibition of hRR lowers dNTPs, which enhances the ability of multiple cancer cell lines, while demonstrating little cytotoxicity gemcitabine triphosphate to be incorporated into growing DNA against normal mobilized peripheral blood progenitor cells. NSAH strands by DNA polymerase. However, gemcitabine causes serious depresses dGTP and dATP levels in the dNTP pool causing S-phase side effects due to its cytotoxicity to normal cells caused by DNA arrest, providing evidence for RR inhibition in cells. This report of a chain termination (20, 21), irreversible inhibition of hRR (22), and nonnucleoside reversible inhibitor binding at the catalytic site of inhibition of numerous other enzymes that recognize phosphate hRRM1 provides a starting point for the design of a unique class moieties, including deoxycytidine deaminase (23), thymidylate of hRR inhibitors. synthase (24), CTP-synthase (25, 26), and topoisomerase 1 (27, 28). ribonucleotide reductase | cancer chemotherapy | small molecule | Significance drug discovery | enzyme regulation The search for anticancer drugs continues to be greatly pursued. Ribonucleotide reductase (RR) is a ubiquitous multisubunit The nucleoside analog gemcitabine, which targets ribonucleotide enzyme that catalyzes the rate-determining step of dNTP reductase (RR) as a diphosphate and DNA polymerases as a tri- synthesis, and its regulation is essential for maintaining a bal- phosphate, is the standard first-line treatment in patients with anced dNTP pool (1). RR is essential for cell growth and genomic pancreatic cancer. However, its cytotoxicity to normal dividing stability, as nucleotide imbalances lead to replication stress and tissues leads to unwanted side effects. Here, we have discovered severe effects on cell growth and survival (2). Therefore, the ac- a nonnucleoside RR inhibitor, naphthyl salicylic acyl hydrazone tivity of RR is tightly regulated at various levels, such as tran- (NSAH), that has efficacy similar to gemcitabine and the poten- scription (3), allostery (1), protein inhibition in Saccharomyces tial to be modified to provide safer and more effective cerevisiae by sml1(4) and cellular localization (5). RR consists of cancer therapies. two subunits: (i) the catalytic subunit called RR1 (α) contains the catalytic or C-site and two allosteric sites, the specificity or S-site Author contributions: M.F.A. and C.G.D. designed research; M.F.A., I.A., S.E.H., J.P., S.A.F., and the activity or A site (Fig. 1A), and (ii) the small subunit and D.S. performed research; M.F.A., T.A.M., N.L.O., W.E.H., and R.V. contributed new β reagents/analytic tools; M.F.A., I.A., S.E.H., J.P., S.A.F., D.S., N.L.O., R.V., and M.E.H. ana- called RR2 ( ) that houses a free radical essential for catalysis lyzed data; and M.F.A., S.E.H., J.P., D.S., M.E.H., and C.G.D. wrote the paper. (6). The catalytic site contains three essential cysteine residues The authors declare no conflict of interest. that conduct thiol-based redox chemistry to reduce the ribose This article is a PNAS Direct Submission. ′ substrate to 2 -deoxyribose (7) (Fig. 1A). In the presence of ef- Data deposition: The atomic coordinates and structure factors have been deposited in the fectors, α exists as a dimer which in the presence of the allosteric Protein Data Bank, www.pdb.org (PDB ID code 5TUS). effectors dATP and ATP form α6β2 and α6βn (n = 2, 4, 6) mul- 1M.F.A., I.A., and S.E.H. contributed equally to this work. – timers, respectively (8 12). Current interest in RR research is 2To whom correspondence should be addressed. Email: [email protected]. increasing our understanding of higher-order oligomerization, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. enzyme turnover, and the mechanism of allosteric regulation that 1073/pnas.1620220114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1620220114 PNAS Early Edition | 1of6 A C-site (NSAH) S-site (TTP) Loop 2 Active site Loop 2 cysteines S-site (TTP) BC nmol dADP Fig. 1. Structural organization of the regulatory subunit hRRM1 and steady-state kinetic studies of NSAH–hRR. (A) A structure of the hRRM1 dimer showing the nonnucleoside small-molecule (magenta) NSAH binding at the C-site. The nucleotide effector TTP is shown as ball and stick (cyan) bound at the S-site, which controls the specificity of the C-site. Active-site cysteine residues are shown in yellow. (B) The specific activity of non–drug-treated hR1 (blue, 162.3 nmol·min−1·mg−1) is comparable to that of the diluted solution of hR1 in the presence of 50 μM NSAH (green, 152.3 nmol·min−1·mg−1). However, an undiluted solution of hR1 in the presence of 50 μM NSAH showed no activity (red). This indicates that NSAH inhibits reversibly. (C) Plot of velocity versus [14C-ADP] for hRRM1 in the presence of 0, 1, 5, 10, 25, and 50 μM NSAH. [14C-ADP] ranged from 0.5 to 5.0 mM. We hypothesize that competitive, reversible, nonnucleoside validated using fluorescence quenching, and in vitro multiple- hRR inhibitors should be less toxic to normal cells, and provide turnover RR kinetic analyses were used to analyze the mode of unique leads for medicinal chemistry and structure-based drug inhibition, as in a similar study (29) (Fig. 1 B and C). hRR enzy- design to advance identification of unique cancer chemothera- matic IC50 values were determined using the method described peutics. Here, we describe the identification and characterization in ref. 29. Six inhibitor concentrations were used ranging from of a hydrazone compound, naphthyl salicylic acyl hydrazone 5 to 100 μM, which yielded an IC50 of 32 ± 10 μM(Fig.