scholarly journals Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule

2017 ◽  
Vol 114 (31) ◽  
pp. 8241-8246 ◽  
Author(s):  
Md. Faiz Ahmad ◽  
Intekhab Alam ◽  
Sarah E. Huff ◽  
John Pink ◽  
Sheryl A. Flanagan ◽  
...  
Keyword(s):  
Small Molecule ◽  
Ribonucleotide Reductase ◽  
Competitive Inhibition ◽  
Large Subunit ◽  
Nucleoside Analogs ◽  
Catalytic Site ◽  
Reversible Inhibitor ◽  
Multiple Cancer ◽  
Dntp Pool ◽  
Starting Point

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.

scholarly journals Clb6-Cdc28 Promotes Ribonucleotide Reductase Subcellular Redistribution during S Phase

2017 ◽  
Vol 38 (6) ◽  
Author(s):  
Xiaorong Wu ◽  
Xiuxiang An ◽  
Caiguo Zhang ◽  
Mingxia Huang
Keyword(s):  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
S Phase ◽  
Nuclear Retention ◽  
Content Type ◽  
Dntp Pool ◽  
Checkpoint Kinases ◽  
Enhanced Sensitivity

ABSTRACTA tightly controlled cellular deoxyribonucleotide (deoxynucleoside triphosphate [dNTP]) pool is critical for maintenance of genome integrity. One mode of dNTP pool regulation is through subcellular localization of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step of dNTP biosynthesis. InSaccharomyces cerevisiae, the RNR small subunit, Rnr2-Rnr4, is localized to the nucleus, whereas the large subunit, Rnr1, is cytoplasmic. As cells enter S phase or encounter DNA damage, Rnr2-Rnr4 relocalizes to the cytoplasm to form an active holoenzyme complex with Rnr1. Although the DNA damage-induced relocalization requires the checkpoint kinases Mec1-Rad53-Dun1, the S-phase-specific redistribution does not. Here, we report that the S-phase cyclin–cyclin-dependent kinase (CDK) complex Clb6-Cdc28 controls Rnr2-Rnr4 relocalization in S phase. Rnr2 contains a consensus CDK site and exhibits Clb6-dependent phosphorylation in S phase. Deletion ofCLB6or removal of the CDK site results in an increased association of Rnr2 with its nuclear anchor Wtm1, nuclear retention of Rnr2-Rnr4, and an enhanced sensitivity to the RNR inhibitor hydroxyurea. Thus, we propose that Rnr2-Rnr4 redistribution in S phase is triggered by Clb6-Cdc28-mediated phosphorylation of Rnr2, which disrupts the Rnr2-Wtm1 interaction and promotes the release of Rnr2-Rnr4 from the nucleus.

scholarly journals Intracellular Internalization and Signaling Pathways Triggered by the Large Subunit of HSV-2 Ribonucleotide Reductase (ICP10)

Virology ◽  
1995 ◽  
Vol 210 (2) ◽  
pp. 345-360 ◽  
Author(s):  
J.C.R. Hunter ◽  
C.C. Smith ◽  
Debashish Bose ◽  
Michael Kulka ◽  
R. Broderick ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Ribonucleotide Reductase ◽  
Large Subunit

scholarly journals Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy

2011 ◽  
Vol 4 (10) ◽  
pp. 1328-1354 ◽  
Author(s):  
Sanath R. Wijerathna ◽  
Md. Faiz Ahmad ◽  
Hai Xu ◽  
James W. Fairman ◽  
Andrew Zhang ◽  
...  
Keyword(s):  
Cancer Chemotherapy ◽  
Ribonucleotide Reductase ◽  
Large Subunit

scholarly journals Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
Author(s):  
Yan Zhang ◽  
Xiuxiang An ◽  
JoAnne Stubbe ◽  
Mingxia Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
Cluster Assembly ◽  
E Coli ◽  
Viability Assay ◽  
Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity

1990 ◽  
Vol 10 (11) ◽  
pp. 5688-5699
Author(s):  
B E Wojcik ◽  
J J Dermody ◽  
H L Ozer ◽  
B Mun ◽  
C K Mathews
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster Ovary ◽  
Reductase Activity ◽  
Chinese Hamster ◽  
Temperature Shift ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Dntp Pool ◽  
Ribonucleotide Reductase Activity

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

scholarly journals 2-Acryloyl-4,5-methylenedioxyphenol: A Small Molecule Endowed with Antidermatophytic Activity

2019 ◽  
Vol 16 (4) ◽  
pp. 461-466
Author(s):  
Marco Zuccolo ◽  
Sabrina Dallavalle ◽  
Raffaella Cincinelli ◽  
Luce Mattio ◽  
Stefania Mazzini ◽  
...  
Keyword(s):  
Small Molecule ◽  
Natural Compound ◽  
Fungal Infections ◽  
Positive Control ◽  
Fungal Species ◽  
Fungal Diseases ◽  
Antifungal Compounds ◽  
Starting Point ◽  
Antidermatophytic Activity

Background: Superficial fungal infections are the most common fungal diseases in humans, affecting more than 25% of the population worldwide. Methods: In the present study, we have investigated the activity of kakuol, a natural compound isolated from the rhizomes of Asarum sieboldii, and some analogues, against various dermatophytes and pharmacologically relevant yeasts. Results: One of the tested compounds, 2-acryloyl-4,5-methylenedioxyphenol, showed a broadspectrum activity against most of the fungal species assayed, resulting particularly effective against dermatophyte strains (MIC values in the range of 0.25-0.5 µg/mL, two/four-fold lower than the positive control miconazole). Conclusion: The results suggest that this molecule can be considered a promising starting point for the development of new antifungal compounds.

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