Glycyrrhetinic acid and its derivatives as inhibitors of poly ( ADP-ribose ) polymerases 1 and 2 , apurinic / apyrimidinic endonuclease 1 and DNA polymerase β

Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences 8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090 1N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences| 9, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090 2UMR7242, University of Strasbourg, CNRS, ESBS Cedex Illkirch Cedex, France, 67412


Introduction.
Alkylating reagents being the oldest class of anticancer drugs are still commonly used; they play an important role in the treatment of several types of cancer.Alkylated bases are predominantly removed by base excision repair (BER) [1].Apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase b (pol b) involved in processing of apurinic/ apyrimidinic sites and DNA synthesis, the common stages of BER independent on type of base damage, can be considered as the most relevant targets.For targeting the whole BER process an alternative approach can be used.It is based on the inhibition of proteins, which regulate the overall efficiency of BER.Poly(ADPribose)polymerases 1 and 2, PARP1 and PARP2, respectively, are considered as regulators of BER [2].Several PARP inhibitors in combination with an alkylating drug, temozolomide, are included in current clinical trials [3].
Thus, specific targeting BER enzymes, catalyzing the key stages, in combination with alkylating reagents is considered as a perspective approach in cancer therapy.Glycyrrhetinic acid (GA) possesses a broad spectrum of pharmacological activities and serves as a base for highly active drug preparations [4,5].GA is an aglycone of glycyrrhizic acid obtained from the roots of licorice plants and represents the main product of its metabolism [4,5].In the two past decades, there has been growing interest in the study of licorice, one of the most ancient medicinal plants that were widely used in Chinese and Tibetian medicine.
The renewed interest in licorice reflects the general trend observed in medicinal practice, where remedies of natural origin are finding increasing application despite considerable success in the use of many synthetic drugs.The drug preparations based on modified natural compounds frequently exceed the parent substances in activity.For instance, several GA derivatives display considerable antitumor activity [6][7][8].
Modulation of BER has the potential to enhance response to chemotherapy and improve outcomes in tumor treatment.In the current study, we aimed to study inhibitory properties of GA derivatives on key BER proteins, PARP1, PARP2, APE1 and pol b, which are considered as targets in cancer therapy.
Physicochemical description (Mp, 1 H and 13 C data) of amides 5-7 are in agreement with literature data [12].
Enzymes and their activity tests.Human PARP1, rat pol b and human APE1 were expressed in Escherichia coli cells and isolated as described in [13][14][15], respectively.Murine PARP2 was expressed in insect cells and purified according to [16].Pol b, PARP1, and PARP2 activity assays were carried out as described [14,17,18], respectively.APE1 activity assay was carried out essentially as described in [15].
Estimation of the inhibitory characteristics of compounds.The inhibitory capacity of the compounds was estimated by comparison of the enzyme activities in specific tests in the presence of compounds at variable concentrations versus their absence.The concentration of potential inhibitors was varied in the range from 100 nM to 1 mM.Depending on properties of tested compounds (solubility in DMSO) and enzyme, we used two types of inhibitory characteristics: residual activity at fixed concentration of compounds or IC 50 values (inhibitor concentration reducing the enzyme activity by half).
Results and discussion.Influence of GA derivatives on poly(ADP-ribose) (PAR) synthesis catalyzed by PARP1 and PARP2.PARP1 and PARP2 are molecular sensors of DNA breaks.Their activity is efficiently stimulated by DNA lesions [19].Involvement of both PARPs in regulation of response to genoxic stress caused by ionizing radiation and alkylating reagents was proved in cells and animal models [19].Catalytic domains of PARP1 and PARP2 display high level of homology and therefore can efficiently recognize the same compounds-inhibitors [20].While, there are structural differences (small insertion in PARP2), which are considered as a basis for creation of selective PARP2 inhibitors [21].
Influence of GA derivatives at different concentration of compounds on PARPs activity was determined at linear part of the dependence of the rate of PAR synthesis versus NAD + concentration.Data for all compounds are summarized in Table and  As whole, GA and its derivatives are inefficient inhibitors of PAR synthesis catalyzed by both PARPs.GA and 7 (morpholine amide of GA) displayed somewhat better inhibition of PARP2 activity.
Influence of GA derivatives on DNA polymerase b activity.Pol b is the main DNA polymerase of base excision repair proceeding via short-and long patch pathways [22][23][24].The level of pol b expression and activity is enhanced in some cancer cells [25,26] that leads to its competition with more accurate replicating DNA polymerases and, as a consequent, to involvement of pol b in extrinsic DNA repair processes, for instance nuc-leotide excision repair [27].Thus pol b inhibitors can provide a therapeutic effect, especially in combination with DNA targeted drugs.
All synthesized GA derivatives were tested as potential pol b inhibitors.A typical curve of the residual pol b activity at variable concentration of 5 is shown in Fig. 3. Data on influence of GA derivatives on pol b activity are summarized in Table.
The ester of GA bearing keto group at the 11 position (ring C), 1 and 2, unlike compounds 3 and 4 with keto group at the 12 position, significantly affect pol b activity.In addition, the inhibitory effect is displayed by pyrrolidine amide 5 and pyperidine amide 6 unlike the morpholine amide 7. It should be noticed that the last compound has a mild inhibitory effect on PARP2 activity.In line with our observation, several triterpenoid derivatives were also shown to inhibit pol b activity with IC 50 being in micromolar range ( [28,29] and references therein).
By and large, all known inhibitors of pol b irrespectively of compound class have IC 50 values in micromolar range [28,29].
Influence of GA derivatives on APE1 activity.Human APE1 is a multifunctional enzyme.APE1 is involved in BER, which eliminates base lesions and spontaneous AP sites being the main AP site hydrolyzing enzyme of higher eukaryotes [30,31].APE1 expression is altered in numerous cancers [31,32].High level of  АРЕ1 protects cells from the action of different genotoxic agents; on the contrary, suppression of APE1 leads to apoptosis and renders cells to become more sensitive to genotoxic agent exposure ( [33] and references therein).Thus АРЕ1 is considered as potential therapeutic target.Selective АРЕ1 inhibitors can be useful both as monotherapy drugs and sensitizers in combined therapy.APE1 inhibitors have demonstrated potentiation of cytotoxicity of alkylating agents in preclinical models [31,[34][35][36].
Representative curve of 3 influence on АРЕ1 activity is shown in Fig. 4. Compound 3 is the only compound, which causes practically full inhibition of APE1 activity at 100 mM concentration.None of other tested compounds influences significantly the AP site hydrolysis (Table ).
IC 50 values of APE1 specific inhibitors discovered to date lie in the submicromolar-low micromolar range [36,37].
Conclusions.The class of GA derivatives, selective pol b inhibitors, was found out.The selective inhibitor of АРЕ1 and weak selective inhibitor of PARP2 were also revealed.
Fig 1.Typical curve for PAR synthesis catalyzed by PARP2 in the presence of GA is shown in Fig. 2.