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Letters in Drug Design & Discovery, 2015, 12, 93-102
Quantum-chemical Study on the Thermodynamical Aspect of Competitive
Inhibition of Ribonucleotide Reductase by trans-Resveratrol, trans-
Piceatannol and Hydroxyurea
Krzysztof Edera,*, Damian Mikulskia,b,* and Marcin Molskib aGen. Zamoyska and Helena Modrzejewska High School no. 2, ul. Matejki 8/10 60-766 Pozna, Poland bDepartment of Theoretical Chemistry, Faculty of Chemistry, A. Mickiewicz University, ul. Umultowska 89B, 61-614 Pozna, Poland Abstract: Ribonucleotide reductase is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleo-
tides. We present for the first time a quantum-chemical study of the thermodynamics of reactions of naturally occurring
potent inhibitors (trans-resveratrol, trans-piceatannol and hydroxyurea) of ribonucleotide reductase with tyrosine radical
and cysteine radical, scavenging of which is crucial for the inhibition of the enzyme. Density functional theory has been
applied to compute the Gibbs free enthalpy changes for these reactions in the gas phase and in the presence of water me-
dium. Various reaction pathways have been analyzed. The results obtained prove that trans-resveratrol 4'-OH group is
mainly responsible for effective reaction with these enzymatic free radicals. In water medium, the reactions studied are
characterized by more negative values of the Gibbs free enthalpy changes than in vacuum. It was found that trans-
resveratrol and trans-piceatannol may be efficient inhibitors of the enzyme (trans-piceatannol is more efficient than trans-
resveratrol). Because inhibition of ribonucleotide reductase is essential for blockage of the cancer development pathways,
the polyphenols studied may block diverse processes (including cancerogenesis) by inhibition of free radical reaction
steps that occur during the catalytic action of this enzyme.
Keywords: Enzyme inhibition, ribonucleotide reductase, trans-resveratrol, trans-piceatannol.
1. INTRODUCTION
carbon atom of ribose – carbon radical is created and it fur-ther induces removal of a hydroxyl anion from the C2' car- Ribonucleoside reductase (RR) (Fig. 2) is an enzymatic
bon atom of ribose. The mechanism of the RR action shows complex that is responsible for the transformation of ribonu- that the tyrosine radical plays a pivotal role in the whole re- cleosides into deoxyribonucleosides and plays a crucial role, action as it works as a catalytic cycle initiator. Therefore, its especially in the process of DNA synthesis [1]. In view of scavenging by antioxidant compounds may lead to an effec- this, the RR has become a target for many anticancer agents tive inhibition of the enzyme [2, 3]. Recent studies have researches and its selective inhibition in cancer tissues is one found that trans-resveratrol and trans-piceatannol, hy- of the main fields the investigation of this enzyme is focused droxyurea (amide form) (Fig. 1) are very active inhibitors of
on. Although in many living organisms this enzyme differs this enzyme [4, 5] and that their activity is caused by the slightly in construction, numerous studies have shown that ability to scavenge tyrosine radical. all of its forms execute similar enzymatic paths and their tertiary structures prove that they are homologous. Structural The main aim of this research is to assess the thermody- investigation has revealed that the enzyme always consists of namical preference of the first step of reaction of competitive two dimeric subsystems: R1 and R2 [1]. inhibition of the ribonucleotide reductase by trans-resveratrol, trans-piceatannol, and hydroxyurea (amide On the basis of a detailed analysis of the mechanism by form), providing additional knowledge about the enzyme which the RR works, we can claim that the catalytic cycle inhibition ability of these compounds. starts from an electron transfer from a cysteine residue to the
tyrosine radical (Fig. 1) which results in the creation of a
very reactive cysteine thiol radical within the subsystem R1.
2. QUANTUM-CHEMICAL COMPUTATIONS
The radical is characterized by the presence of an unpaired All quantum calculations were performed within the electron on a sulfur atom [1] - its very high reactivity is GAUSSIAN 09 computational package [6]. The total mo- caused by the lack of possibility of resonance stabilization. lecular energy calculations and structure optimizations have The radical then detaches a hydrogen atom from the C3' been carried out for the chemical systems considered in vac-uum and in the presence of water medium without symmetry constraints in the ground state. In computations, the amide *Address correspondence to this author at the Department of Theoretical form of hydroxyurea was used since in the media studied it Chemistry, Faculty of Chemistry, A. Mickiewicz University, ul. Umul- is its most energetically stable tautomeric form (each towska 89B, 61-614 Pozna, Poland; Tel: +48504522243, +48609726081; tautomeric form of the hydroxyurea had been fully optimized E-mails: [email protected], [email protected] at the same level as other compounds and the most energeti- ;/15 $58.00+.00
2015 Bentham Science Publishers
94 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2
Eder et al.
analysis at the same level of theory was used to verify if the structure corresponds to a stationary point on the potential energy surface. Thermodynamical preference of the reactions of electron transfer and hydrogen transfer from t-RES to tyrosine radical was determined by means of the Gibbs free enthalpy changes of the reactions. The same parameters were computed for the reaction of hydroxyurea with tyrosine radi-cal in order to compare the inhibition potential of t-RES and hydroxyurea and for the reactions of t-RES and hydroxyurea with cysteine thiol radical with the latter being created dur- ing the catalytic cycle. The Gibbs free enthalpy change of the hydroxyurea (amid form) reaction of t-RES with tyrosine anion radical was computed Fig. (1). Molecular structures of the trans-resveratrol, trans-
as the tyrosine (with pI = 5.64 ) occurs mainly in the anion piceatannol, tyrosine radical and hydroxyurea (amide form). form (deprotonated carboxyl group) in neutral pH. Gibbs free enthalpy changes of the reactions were computed as the Gibbs free enthalpy differences of products and reactants under standard conditions T = 298,15K; p = 1,00105 Pa solvation effects were considered by means of the Conduc-tor-like Polarizable Continuum Model (CPCM) that is based on the COSMO model [8] implemented in GAUSSIAN 09 package. The CPCM computations were performed with tesserae of 0.2  average size. The polarizable dielectric medium is described by a dielectric constant of the solvent (78.39 for water). The solvation effects on the values of the Gibbs free enthalpy changes of the reactions were estimated for the vacuum equilibrium geometries. 3. RESULTS AND DISCUSSION
All of the chemical reactions investigated are presented in Figs. (3, 4 and 5). The corresponding Gibbs free enthalpy
changes of the reactions for vacuum and water medium are
shown consecutively in Tables 1, 2 and 3.
Fig. (2). The molecular structure of the ribonucleotide reductase
All of the first-step reactions of ribonucleotide reductase competitive inhibition were found to be more preferred thermodynamically (to have lower Gibbs free enthalpy cally stable form had been chosen). For the optimization of change magnitude) in water medium than in vacuum. Each geometries and energies of t-RES (trans-resveratrol), t-RES chemical reaction of the potential inhibitor with tyrosine or phenoxyradical, t-PIC (trans-piceatannol), hydroxyurea, cysteine radical, the products of which are inhibitor radical hydroxyurea oxyradical, tyrosine, tyrosine radical, tyrosine and neutral molecule of tyrosine or cysteine correspondingly anion, tyrosine anion radical, cysteine and cysteine thiol (Reactions R1, R2, H1, P1, P2, P3; Figs. (3, 4 and 5)), is
radical in their ground states, the restricted Becke's three- fully allowed in the thermodynamical sense. It is important, parameter hybrid functional B3LYP with the gradient- that the reaction of hydroxyurea has the lowest and the reac- corrected correlation functional by Lee et al. [7] and 6- tions of trans-resveratrol have the highest Gibbs free en- 311++G(3df,2p) basis-set were employed. To confirm the thalpy change from among the reactions proceeding by this positions of the minima, geometry optimization was per- pathway. We claim that the relatively small size of the hy- formed, starting from the most stable conformations obtained droxyurea molecule and the presence of the hydroxyamide from the potential energy surface (PES). Potential energy group are the two main factors that are responsible for the surfaces were built by changing the dihedral angles  (C5-C4- lower free enthalpy change of the hydroxyurea reaction with C7-C8) and  (C7-C8-C9-C10) in trans-resveratrol and trans- tyrosine radical. On the basis of this we can claim that hy- piceatannol. The conformational energy maps were obtained droxyurea is a stronger inhibitor of the ribonucleotide reduc- through discrete rotation of these selected dihedral angles, in tase than trans-resveratrol and trans-piceatannol, whilst 10° increments from 0° to 180°. At each point, the total en- trans-piceatannol has higher inhibiting potential than trans- ergy of t-RES and t-PIC was computed at the B3LYP/6- 311++G(3df,2p) level of theory. Afterwards, the most stable structures obtained from the energy profiles were fully opti- On the other hand, most of the reactions in which the ty- mized without any geometrical and symmetry constraints rosine anion radical takes place are not preferred (they are around each potential minimum. This computational proce- preferable when hydroxyurea takes part in the reaction (Re-
dure has been employed in order to obtain the equilibrium action H2) and not preferable when trans-resveratrol (Reac-
structures of the chemical systems studied in their energy tions R3 and R4) or trans-picetannol (Reactions R3, R4 and
minima. For each fully optimized structure, the frequency R5) takes part). This difference in the Gibbs free enthalpy
Quantum-chemical Study on the Thermodynamical Aspect
Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2 95
The respective Gibbs free enthalpy changes
The respective Gibbs free enthalpy changes
[kcal*mol-1] of the possible first-step competitive inhi-
[kcal*mol-1] of the possible first-step competitive inhi-
bition reactions of the RR by trans-resveratrol and the
bition reactions of the RR by the trans-piceatannol
reaction of deprotonation of the t-RES (Reaction R6)
and the reaction of deprotonation of the t-PIC (Reac-
presented in the Fig. (3) obtained at the B3LYP/6-
tion P8) presented in the Fig. (5) obtained at the
311++G(3df,2p) level of theory in vacuum and water
B3LYP/6-311++G(3df,2p) level of theory in vacuum
and water medium.
Vacuum Gibbs Free
Water Medium Gibbs
Vacuum Gibbs Free
Water Medium Gibbs
Reaction
Enthalpy Change
Free Enthalpy Change
Free Enthalpy Change
The respective Gibbs free enthalpy changes
[kcal*mol-1] of the possible first-step competitive inhi-
bition reactions of the RR by the amide form of the
hydroxyurea and the reaction of deprotonation of the
hydroxyurea (Reaction H4) presented in the Fig. (4)
the proton from the tyrosine to the inhibitor is not preferred. obtained at the B3LYP/6-311++G(3df,2p) level of the-
All reactions of the tyrosine and cysteine thiol radicals that ory in vacuum and water medium.
lead to creation of inhibitor radical (not inhibitor cation radi-cal) are fully preferred thermodynamically. Vacuum Gibbs Free
Water Medium Gibbs
Reaction
Reactions R6, H4 and P8 of deprotonation of the inhibi-
Enthalpy Change
Free Enthalpy Change
tors studied are fully allowed thermodynamically which -15.679 means that they proceed spontaneously. The reactions of the 4'-OH group of t-RES and t-PIC with the tyrosine radical are preferred thermodynamically 1.479 rather than the reactions in which the 3-OH group takes part. -8.210 Moreover, also the reactions of these inhibitors with tyrosine anion radical are more preferred when in their proceeding the -15.987 unpaired electron is localized on the 4'-O atom than when it is created on the 3-O atom (even though these reactions are change can be caused by the distinction between stabilities not thermodynamically allowed) – (compare the Gibbs free of tyrosine radical and tyrosine anion radical. In the first case enthalpy changes of the Reactions R3 and R4 and those of
the species are only stabilized by delocalization of the Reactions P4 and P6). This is caused by a higher reso-
the unpaired electron over the phenyl ring (resonance nance stabilization of the 4'-O-radical than 3-O-radical. For stabilization). In the second case the species are additionally this type of reactions in which t-PIC takes part, the most sta- stabilized by delocalization of the negative charge over the ble radical is the one with the unpaired electron localized on carboxyl group. Therefore, the free energy of the reactants is the 4'-O atom and the least stable – the one with the un- lower in the reactions of tyrosine anion radical and the Gibbs paired electron localized on the 3'-O atom (Reactions P4-
free enthalpy change is higher for those reactions. That is the P6). Also this result is provided by diverse extent of reso-
reason why they are not preferred thermodynamically. nance stabilization in different unpaired electron locations in Additionally, we can notice that all of the reactions of ty- radicals. t-PIC reactions result in the creation of inhibitor rosine radical in which cation radicals of potential inhibitors cation radicals because Gibbs free enthalpy change magni- are formed are not thermodynamically preferred in the media tudes are in opposition to those obtained from the analysis of studied (apart from the Reaction P7 of trans-piceatannol
the reactions that result in the creation of inhibitor radicals. It with tyrosine radical in the water medium). We claim that was found that in the latter case the compound in which the this can be caused by a higher acidity of the inhibitors stud- unpaired electron is localized on the 3'-O atom is more ied than tyrosine radical – that is the reason why transfer of stable than the one in which it is localized on the 3-O atom. 96 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2
Eder et al.
Fig. (3). Contd….
Quantum-chemical Study on the Thermodynamical Aspect
Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2 97
Fig. (3). The schemes of the possible first-step competitive inhibition reactions of the RR by trans-resveratrol and the reaction of deprotona-
tion of the t-RES (reaction R7).
98 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2
Eder et al.
Fig. (4). Contd….
Quantum-chemical Study on the Thermodynamical Aspect
Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2 99
Fig. (4). The schemes of the possible first-step competitive inhibition reactions of the RR by the amid form of the hydroxyurea and the reac-
tion of deprotonation of the hydroxyurea (reaction H5).
Fig. (5). Contd….
100 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2
Eder et al.
Fig. (5). Contd….
Quantum-chemical Study on the Thermodynamical Aspect
Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2 101
Fig. (5). The schemes of the possible first-step competitive inhibition reactions of the RR by the trans-piceatannol and the reaction of depro-
tonation of the t-PIC (reaction P7).
102 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 2
Eder et al.
As it is not associated with any chemical justification we claim that this small difference is caused by the specifics of The calculations were performed at the Pozna the quantum method applied. Supercomputing and Networking Center (PCSS). We grate- The reactions of t-RES and t-PIC with tyrosine radical fully acknowledge the support of this work by the grant no. and cysteine thiol radical are characterized by similar magni- 182, "Theoretical study of the anticancer activity of naturally tudes of the free enthalpy change which are negative. These occurring compounds and their analogs". results suggest that t-RES and t-PIC can effectively scavenge both tyrosine radical and cysteine thiol radical, and therefore are strong chemotherapeutic compounds, which effectively REFERENCES
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CONFLICT OF INTEREST
The authors confirm that this article content has no con- flict of interest. Received: July 07, 2014 Revised: August 31, 2014 Accepted: September 07 2014

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