The Structural Basis for Peptidomimetic Inhibition of Eukaryotic Ribonucleotide Reductase: A Conformationally Flexible Pharmacophore

Xu, Hai; Fairman, J.W.; Wijerathna, Sanath R.; Kreischer, N.R.; LaMacchia, J.; Helmbrecht, Elizabeth; Cooperman, Barry S.; Dealwis, Chris

doi:10.1021/jm800350u

Cited by 22 publications

(11 citation statements)

References 29 publications

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“…36 The soaking conditions were partly guided by our ITC data. Instead of using extremely saturating nucleotide concentrations as previously used, 23 we used 0.5 mM ADP in our soaking experiments.…”

Section: Methodsmentioning

confidence: 99%

Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

Ahmad

Kaushal

Wan

et al. 2012

Journal of Molecular Biology

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Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (α) that contains the catalytic site and RR2 (β) that houses a diferrictyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP–CDP [AMPPNP, adenosine 5-(β,γ-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.

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Section: Methodsmentioning

confidence: 99%

Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

Ahmad

Kaushal

Wan

et al. 2012

Journal of Molecular Biology

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“…P7 and P6, both of which inhibit ScRR. 25) So the subsite B (PDB id-2ZLF) was selected as the receptor. The interior of the cavity in subsite B was narrow and consisted of two pockets accommodating the hydrophobic residues.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Anticancer Evaluation of Benzyloxyurea Derivatives

Ren

Zhong

Mai

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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A series of novel benzyloxyurea derivatives was designed, synthesized by substituting different benzyls or phenyls on N,N′-positions of the hydroxyurea (HU). These target compounds were evaluated for their anticancer activity in vitro against human leukemia cell line K562 and murine leukemia cell line L1210 in comparison with HU by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Some of the compounds showed promising anticancer activity against the cells. Molecular docking experiments with Saccharomyces cerevisiae R1 domain indicated that 4a and 4f′ have stronger affinity than 4m and 4n. Flow cytometry study showed that compound 4g exerted greater apoptotic activity against K562 cells line than HU.Key words benzyloxyurea derivative; anticancer evaluation; K562; L1210; molecular docking; apoptosis As we all know, because of its low cure rate and high mortality, tumor has become one of the most terrible diseases around the world. Hydroxyurea (HU), as the preferred treatment drug of chronic myelogenous leukemia, could also be used to treat melanoma, tumor of the ovary, human immunodeficiency virus (HIV) infection, β-mediterranean disease and so on. [1][2][3][4][5] But the use of HU is limited by its metabolic and inherent cytotoxicity. [6][7][8][9] Eukaryotic ribonucleoside reductase (RR) catalyzes nucleoside diphosphate conversion to deoxynucleoside diphosphate. Crucial for rapidly dividing cells, RR is an attractive therapeutic target of cancer, and over the past years, many chemotherapeutics inhibiting different subunits of RR have been developed and tested clinically for their anticancer activities and anti-HIV activities.10-13) HU is the first RR inhibitor applied to the clinic, and is utilized for the therapeutic of neoplasms because of its influences on the DNA replication of cancer cells. 1,14) To overcome the drawbacks of HU, numerous medicinal chemistry efforts have been made to design and synthesize novel HU derivatives. 9,[15][16][17][18] In previous paper, we reported HU monosubstituents and disubstituents with benzyls at N-positions of HU. 6,19) The results indicated that the stronger hydrophobic nature of the HU derivatives might favor the cytotoxic activity and benzyl groups at the N-position of HU were associated with enhanced cytotoxic activity. The disadvantages associated with HU's physicochemical properties, e.g., very high hydrophilicity (log P: −1.80) and small molecular size, may be overcomed by the structural modification. This background prompted us to further explore more novel benzyloxyurea derivatives with different substituents at N,N′-positions of HU. In this study, a series of novel benzyloxyurea derivatives substituted with different benzyls or phenyls at N,N′-position of HU were synthesized. The target compounds were evaluated for their anticancer activity in vitro against human leukemia cell line K562 and murine leukemia cell line L1210 in comparison with HU by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Meanwhile, the...

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“…[10] Rnr1p was expressed in BL21 (DE3)plys cells at 15°C using IPTG. Bacterial cells were harvested and proteins were precipitated in 40% ammonium sulfate.…”

Section: Methodsmentioning

confidence: 99%

Structure‐Based Design, Synthesis, and Evaluation of 2′‐(2‐Hydroxyethyl)‐2′‐deoxyadenosine and the 5′‐Diphosphate Derivative as Ribonucleotide Reductase Inhibitors

Sun

Wijerathna

et al. 2009

ChemMedChem

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Analysis of the recently solved X-ray crystal structures of yeast ribonucleotide reductase I (RnrI) in complex with effectors and substrates led to the discovery of a conserved water molecule located at the active site that interacted with the 2′ hydroxy of the nucleoside ribose. In this study 2′-(2-hydroxyethyl)-2′-deoxy-adenosine 1 and its 5′-diphosphate 2 were designed and synthesized to see if the conserved water molecule could be displaced by a hydroxylmethylene group, to generate a novel of inhibitors of this enzyme towards the development of potential anti-neoplastic agents. In this paper, we report the synthesis of these two adenosine analogs 1 and 2, and the cocrystal structure of adenosine diphosphate analog 2 bound with RnrI enzyme which displaces the conserved water as hypothesized.

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The Structural Basis for Peptidomimetic Inhibition of Eukaryotic Ribonucleotide Reductase: A Conformationally Flexible Pharmacophore

Cited by 22 publications

References 29 publications

Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

Synthesis and Anticancer Evaluation of Benzyloxyurea Derivatives

Structure‐Based Design, Synthesis, and Evaluation of 2′‐(2‐Hydroxyethyl)‐2′‐deoxyadenosine and the 5′‐Diphosphate Derivative as Ribonucleotide Reductase Inhibitors

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