Marta Hammerstad

New Iminodiacetate–Thiosemicarbazone Hybrids and Their Copper(II) Complexes Are Potential Ribonucleotide Reductase R2 Inhibitors with High Antiproliferative Activity

As ribonucleotide reductase (RNR) plays a crucial role in nucleic acid metabolism, it is an important target for anticancer therapy. The thiosemicarbazone Triapine is an efficient R2 inhibitor, which has entered ∼20 clinical trials. Thiosemicarbazones are supposed to exert their biological effects through effectively binding transition-metal ions. In this study, six iminodiacetate-thiosemicarbazones able to form transition-metal complexes, as well as six dicopper(II) complexes, were synthesized and fully characterized by analytical, spectroscopic techniques (IR, UV-vis; 1H and 13C NMR), electrospray ionization mass spectrometry, and X-ray diffraction. The antiproliferative effects were examined in several human cancer and one noncancerous cell lines. Several of the compounds showed high cytotoxicity and marked selectivity for cancer cells. On the basis of this, and on molecular docking calculations one lead dicopper(II) complex and one thiosemicarbazone were chosen for in vitro analysis as potential R2 inhibitors. Their interaction with R2 and effect on the Fe(III)2-Y· cofactor were characterized by microscale thermophoresis, and two spectroscopic techniques, namely, electron paramagnetic resonance and UV-vis spectroscopy. Our findings suggest that several of the synthesized proligands and copper(II) complexes are effective antiproliferative agents in several cancer cell lines, targeting RNR, which deserve further investigation as potential anticancer drugs.

Tuning of thioredoxin redox properties by intramolecular hydrogen bonds.

Thioredoxin-like proteins contain a characteristic C-x-x-C active site motif and are involved in a large number of biological processes ranging from electron transfer, cellular redox level maintenance, and regulation of cellular processes. The mechanism for deprotonation of the buried C-terminal active site cysteine in thioredoxin, necessary for dissociation of the mixed-disulfide intermediate that occurs under thiol/disulfide mediated electron transfer, is not well understood for all thioredoxin superfamily members. Here we have characterized a 8.7 kD thioredoxin (BC3987) from Bacillus cereus that unlike the typical thioredoxin appears to use the conserved Thr8 side chain near the unusual C-P-P-C active site to increase enzymatic activity by forming a hydrogen bond to the buried cysteine. Our hypothesis is based on biochemical assays and thiolate pKa titrations where the wild type and T8A mutant are compared, phylogenetic analysis of related thioredoxins, and QM/MM calculations with the BC3987 crystal structure as a precursor for modeling of reduced active sites. We suggest that our model applies to other thioredoxin subclasses with similar active site arrangements.

Crystal Structure of Bacillus Cereus Class Ib Ribonucleotide Reductase Di-Iron Nrdf in Complex with Nrdi.

Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (β2) subunit to initiate ribonucleotide reduction in the NrdE (α2) subunit. Contrary to the diferric tyrosyl radical (Fe(III)2-Y•) cofactor, which can self-assemble from Fe(II)2-NrdF and O2, generation of the Mn(III)2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-Mn(II)2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein-protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus Mn(II)2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the dimetal site and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF dimetal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested α2β2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus.

Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. In Bacillus cereus there are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5 Å resolution from crystals belonging to the orthorhombic space group P2₁2₁2, with unit-cell parameters a=57.2, b=164.3, c=95.0 Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.

Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus

To reduce ribonucleotides to deoxyribonucleotides, the manganese-bound form of class Ib ribonucleotide reductase (RNR) must be activated via a pathway that involves redox protein(s). The reduced flavoprotein NrdI is an important protein in this pathway, as it reduces dioxygen to superoxide. Superoxide then reacts with the RNR Mn(II)2 site to generate a tyrosyl radical that is required for catalysis. A native NrdI reductase has not yet been identified. We herein demonstrate through kinetic and spectroscopic studies that an endogenous flavodoxin reductase can function as the NrdI reductase in Bacillus cereus. When the flavodoxin reductase reduces NrdI, tyrosyl radical formation in RNR is promoted under aerobic conditions, significantly increasing the radical yield. Thus, a missing piece of the class Ib RNR NrdI redox pathway has finally been identified.

Nitric oxide synthase and possible redox partners in Bacillus cereus

Nitric oxide synthase (NOS), a BH4-dependent heme-enzyme, is the only enzyme that specifically produces NO in mammals. NO is produced by the NOS homodimer in two multistep reaction cycles involving electron transfer from a reducing domain to the heme active site. The importance of NO in mammals is due to its function in signalling, vasodilation and immune response. Some bacterial species also contain NOS-encoding genes, but these bacterial NOSs are differently organized – they contain no reducing domain – and their functions and mechanism are not fully resolved [1]. Bacterial NOSs are potential drug targets, because of their role in protection against antibiotics and oxidative stress in some pathogenic bacterial species (e.g. Bacillus anthracis) [2]. Flavodoxins (Flds) have been shown to be relevant redox partners for bacterial NOSs [3], but the specificity of the interaction between NOS and Flds remains poorly understood. We have investigated the NOS protein system in Bacillus cereus, whose genome encodes NOS and two Flds, by combining crystallographic and spectroscopic methods. So far the structures of the two Flds have been solved to 0.98 Å and 2.75 Å resolution, while NOS has been solved to 2.9 Å resolution. An important part of the study has been to investigate the effect of synchrotron X-ray radiation on the oxidation state and structure of the Flds, due to their radiation sensitive cofactor flavin mononucleotide (FMN). The high-resolution (0.98 Å), oxidized structure of one Fld indicates that X-rays induce structural changes around the FMN cofactor. Another important part of the study has been to gain further insight into the specificity and flexibility of the interactions between ferredoxin/flavodoxin-NADP+ reductases, Flds and NOS in Bacillus cereus, as well as the possible mechanism of bacterial NOSs.

Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex

Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (beta2) subunit to initiate ribonucleotide reduction in the NrdE (beta2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y•) cofactor, which can selfassemble from FeII2-NrdF and O2, generation of the MnIII2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein-protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus MnII2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the di-metal site, and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF di-metal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested alfa2beta2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved. [1]

Correction: Tuning of Thioredoxin Redox Properties by Intramolecular Hydrogen Bonds

Figures 1 and 2 were accidentally switched. The image appearing as Figure 1 belongs with the title and legend for Figure 2, and the image appearing as Figure 2 belongs with the title and legend for Figure 1. The titles and legends are in correct order.