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Activation and inhibition of diiron and iron-manganese ribonucleotide reductases
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
2012 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Ribonucleotide reductase (RNR) catalyses the reduction of ribonucleotides to deoxyribonucleotides. In conventional class I RNRs the active site is located in the R1 subunit, and the R2 subunit contains a diiron cofactor and a stable tyrosyl radical essential for activity.

Class Ic Chlamydia trachomatis RNR lacks the tyrosyl radical and uses a Mn(IV)Fe(III) cofactor for catalysis. The requirement for metals for RNR activation was studied in C. trachomatis F127Y and Y129F R2, and in mouse wild type and Y177F R2 proteins. The results indicate that R2 affinity for metals is determined by the amino acid located next to the metal site, at the position of the radical carrying tyrosyl. Specifically, R2 proteins that contain phenylalanine in this place bind Mn and Fe, and the tyrosyl containing R2s bind only Fe.

Further results show that C. trachomatis RNR can be inhibited by R2 C-terminal oligopeptides. The highest inhibition was observed for a 20-mer peptide indicating that the oligopeptide inhibition mechanism of class Ic is similar to that of the class Ia and b.

The second part of the thesis deals with class Ia RNR inhibition. The results show that a lanthanum complex containing three 1,10-phenantroline molecules (KP772) which has showed promising cytotoxic activity in cancer cell lines inhibits mouse R2 protein in the presence of external reductants by iron-chelation. It is suggested that KP772 has several synergistic inhibition mechanisms that contribute to its overall anticancer activity. Moreover, other results show that Triapine, a promising chemotherapeutic compound, and its Fe, Ga, Zn, and Cu complexes, inhibit mouse R2 in both reducing and non-reducing conditions. Inhibition by Triapine involves the binding of the drug to the surface of the R2 protein leading to labilization of the Fe-center and subsequent Fe-chelation by Triapine. Formation of the Fe(II)-Triapine complex which reacts with O2 to produce reactive oxygen species results in complete RNR inactivation.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2012. , s. 64
Nyckelord [en]
class Ic, C. trachomatis, cytotoxicity, KP772, lanthanum, manganese–iron cofactor, metal complex, oligopeptide inhibitor, ribonucleotide reductase, Triapine, tyrosyl radical
Nationell ämneskategori
Biofysik
Forskningsämne
biofysik
Identifikatorer
URN: urn:nbn:se:su:diva-75175ISBN: 978-91-7447-499-2 (tryckt)OAI: oai:DiVA.org:su-75175DiVA, id: diva2:514827
Disputation
2012-05-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2012-05-09 Skapad: 2012-04-11 Senast uppdaterad: 2012-04-16Bibliografiskt granskad
Delarbeten
1. Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study
Öppna denna publikation i ny flik eller fönster >>Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study
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2011 (Engelska)Ingår i: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948, Vol. 105, nr 11, s. 1422-1431Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.

Ort, förlag, år, upplaga, sidor
Elsevier, 2011
Nyckelord
Ribonucleotide reductase (RNR), Triapine, Tyrosyl radical, Metal complex, Cytotoxicity, EPR
Nationell ämneskategori
Organisk kemi Biofysik
Forskningsämne
biofysik
Identifikatorer
urn:nbn:se:su:diva-69327 (URN)10.1016/j.jinorgbio.2011.07.003 (DOI)000297565500008 ()
Forskningsfinansiär
Vetenskapsrådet
Anmärkning
8Tillgänglig från: 2012-01-11 Skapad: 2012-01-11 Senast uppdaterad: 2019-12-09Bibliografiskt granskad
2. Ribonucleotide Reductase as One Important Target of [Tris(1,10- phenanthroline)lanthanum(III)] Trithiocyanate (KP772)
Öppna denna publikation i ny flik eller fönster >>Ribonucleotide Reductase as One Important Target of [Tris(1,10- phenanthroline)lanthanum(III)] Trithiocyanate (KP772)
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2009 (Engelska)Ingår i: Current Cancer Drug Targets, ISSN 1568-0096, E-ISSN 1873-5576, Vol. 9, nr 5, s. 595-607Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

KP772 is a new lanthanum complex containing three 1,10-phenathroline molecules. Recently, we have demonstrated that the promising in vitro and in vivo anticancer properties of KP772 are based on p53-independent G(0)G(1) arrest and apoptosis induction. A National Cancer Institute (NCI) screen revealed significant correlation of KP772 activity with that of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU). Consequently, this study aimed to investigate whether KP772 targets DNA synthesis in tumor cells by RR inhibition. Indeed, KP772 treatment led to significant reduction of cytidine incorporation paralleled by a decrease of deoxynucleoside triphosphate (dNTP) pools. This strongly indicates disruption of RR activity. Moreover, KP772 protected against oxidative stress, suggesting that this drug might interfere with RR by interaction with the tyrosyl radical in subunit R2. Additionally, several observations (e.g. increase of transferrin receptor expression and protective effect of iron preloading) indicate that KP772 interferes with cellular iron homeostasis. Accordingly, co-incubation of Fe(II) with KP772 led to generation of a coloured iron complex (Fe-KP772) in cell free systems. In electron paramagnetic resonance (EPR) measurements of mouse R2 subunits, KP772 disrupted the tyrosyl radical while Fe-KP772 had no significant effects. Moreover, coincubation of KP772 with iron-loaded R2 led to formation of Fe-KP772 suggesting chelation of RR-bound Fe(II). Summarizing, our data prove that KP772 inhibits RR by targeting the iron centre of the R2 subunit. As also Fe-KP772 as well as free lanthanum exert significant -though less pronounced- cytotoxic/static activities, additional mechanisms are likely to synergise with RR inhibition in the promising anticancer activity of KP772.

Nyckelord
Lanthanum, ribonucleotide reductase, 1, 10-phenanthroline, cell cycle arrest, anticancer activity, KP772
Nationell ämneskategori
Biofysik
Forskningsämne
biofysik; biokemi
Identifikatorer
urn:nbn:se:su:diva-31508 (URN)10.2174/156800909789056962 (DOI)000269238600001 ()19508176 (PubMedID)
Tillgänglig från: 2009-11-18 Skapad: 2009-11-18 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
3. Metal binding and activity of ribonucleotide reductase protein R2 mutants: Conditions for formation of the mixed manganese-iron cofactor
Öppna denna publikation i ny flik eller fönster >>Metal binding and activity of ribonucleotide reductase protein R2 mutants: Conditions for formation of the mixed manganese-iron cofactor
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2009 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 27, s. 6532-6539Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

Nyckelord
metal, activity, R2 protein, mutant, manganese-iron cofactor
Nationell ämneskategori
Biofysik
Forskningsämne
biofysik; biokemi
Identifikatorer
urn:nbn:se:su:diva-31507 (URN)10.1021/bi900693s (DOI)000268137600028 ()19492792 (PubMedID)
Tillgänglig från: 2009-11-18 Skapad: 2009-11-18 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
4. Inhibition of chlamydial class Ic ribonucleotide reductase by C-terminal peptides from protein R2
Öppna denna publikation i ny flik eller fönster >>Inhibition of chlamydial class Ic ribonucleotide reductase by C-terminal peptides from protein R2
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2011 (Engelska)Ingår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 17, nr 11, s. 756-762Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese-iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C-terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical-containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese-iron cofactor is similar to that of the tyrosyl-radical-containing RNRs, involving reversible long-range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2-derived oligopeptide blocks the transfer pathway. We have constructed three-dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20-mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C-terminus of protein R2.

Nyckelord
class Ic ribonucleotide reductase, manganese–iron cofactor, subunit interaction inhibitor, protein R2 C-terminus, oligopeptide inhibitor
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biofysik
Identifikatorer
urn:nbn:se:su:diva-70146 (URN)10.1002/psc.1399 (DOI)000296493600007 ()21976435 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet
Anmärkning
authorCount>6Tillgänglig från: 2012-01-17 Skapad: 2012-01-17 Senast uppdaterad: 2017-12-08Bibliografiskt granskad

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