Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): a combined experimental and theoretical study

Abstract

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.

Fig. 1

Concentration distribution curves of (a) 1(Fe) and 2(Ga) (the gallium complex is completely dissociated in the whole concentration range) and (b) 3(Zn) and 4(Cu) in relation to the concentration of complex at pH 7.60. Calculations are based on data reported in [23,24].

Fig. 2

UV/Vis absorption spectra of HL and its metal complexes 1(Fe), 2(Ga), 3(Zn) and 4(Cu), under a) non-reducing, and b) reducing conditions. Samples contained 25 μM HL or 50 μM complex in 1% (w/w) DMSO/H₂O, Tris buffer, pH 7.60, at 295 K and 2 mM DTT (only b).

Fig. 3

a) Tyrosyl radical destruction in mouse R2 RNR protein by HL and its metal complexes, 1(Fe), 2(Ga), 3(Zn) and 4(Cu), in absence of DTT. Samples containing 30 μM mouse R2 protein and 30 μM compound (1% (w/w) DMSO/H₂O) in Tris buffer, pH 7.60, were incubated for indicated times and quickly frozen in cold isopentane. The natural decay of tyrosyl radical in the R2 protein was subtracted for each point (see Supplementary Data). b) UV/Vis spectra of the same samples as in (a) taken after 15 min reaction time at 295 K. The spectrum of 30 μM HL without R2 RNR protein (– – –) is shown for comparison.

Fig. 4

a) Tyrosyl radical destruction in mouse R2 RNR protein by HL and its metal complexes, 1(Fe), 2(Ga), 3(Zn) and 4(Cu), in the presence of DTT. Samples containing 30 μM mouse R2 protein, 2 mM DTT, and 30 μM compound (1% (w/w) DMSO/H₂O) in Tris buffer, pH 7.60, were incubated for indicated times and quickly frozen in cold isopentane. The decay of the tyrosyl radical in the R2 protein in the presence of 2 mM DTT was subtracted for each point (see Supplementary Data). b) UV/Vis spectra of the same samples as in (a) taken after 5 min reaction time at 295 K.

Fig. 5

Tyrosyl radical destruction in mouse R2 RNR protein by HL and its metal complexes, 1(Fe), 2(Ga), 3(Zn) and 4(Cu) in a) absence and b) presence of DTT. Samples containing 30 μM mouse R2 protein and 6 μM compound (1% (w/w) DMSO/H₂O) in Tris buffer, pH 7.60, and 2 mM DTT (b only) were incubated for indicated times and quickly frozen in cold isopentane. The natural decay of tyrosyl radical in mouse R2 protein without DTT (a) or in the presence of DTT (b) was subtracted for each point.

Fig. 6

Tyrosyl radical destruction in mouse R2 RNR protein by compound 1(Fe) ina) absence and b) presence of DTT. Samples containing 30 μM mouse R2 protein and 30, 6 or 0.125 μM compound 1(Fe) (1% (w/w) DMSO/H₂O) in Tris buffer, pH 7.60, and 2 mM DTT (b only) were incubated for indicated times and quickly frozen in cold isopentane. The top curve in a) was obtained in anaerobic conditions (oxygen was removed from the protein by gentle evacuation and refilling with argon over a period of 20 min). The natural decay of tyrosyl radical in mouse R2 protein in the absence (a) or in the presence of DTT (b) was subtracted for each point.

Fig. 7

The calculated binding pocket for Triapine on the surface of the mouse R2 RNR protein. a) Structure of mouse R2 RNR protein with bound Triapine. The red and blue areas represent positive and negative charge, respectively, in the surface potential map. Triapine is shown in stick representation (cyan). b) Binding site showing hydrogen bonding between Triapine (cyan) and Glu233, Glu335, Asp272 and Arg331; and interactions with Phe237, Phe241, Ser238 and Tyr324. All residues are shown in black, only Glu233 in magenta, for clarity. The diferric center is represented as two orange spheres.

Scheme 1

Chemical structures of Triapine and the metal complexes investigated in this study.

Scheme 2

Summary of possible mechanism steps of mouse RNR inhibition by Triapine under non-reducing and reducing conditions. Less efficient inhibition, observed in non-reducing conditions, involves binding of Triapine to mouse R2 RNR protein which leads to facillitated release of iron(III) from the protein. Subsequently, Triapine chelates iron(III) to form [Fe(III)L₂]⁺. In the presence of mouse R2 protein the iron complex is reduced to [Fe(II)L₂], which then in presence of molecular oxygen leads to ROS induced inactivation of the R2 protein. More efficient inhibition, observed under reducing conditions, involves the reduction of the diferric center in mouse R2 protein by the external reductant DTT, followed by chelation of released iron(II) by HL. The formed [Fe(II)L₂] complex activates molecular oxygen and results in ROS induced inactivation of the R2 protein. Reaction steps that take place in the absence or presence of external reductant are shown in white and light gray, respectively; Reaction step that requires the presence of oxygen is shown in dark gray.