A type I combi-targeting approach for the design of molecules with enhanced potency against BRCA1/2 mutant- and O6-methylguanine-DNA methyltransferase (mgmt)- expressing tumour cells

Abstract

Background: Mutations of the DNA repair proteins BRCA1/2 are synthetically lethal with the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), which when inhibited, leads to cell death due to the absence of compensatory DNA repair mechanism. The potency of PARP inhibitors has now been clinically proven. However, disappointingly, acquired resistance mediated by the reactivation of wild type BRCA1/2 has been reported. In order to improve their efficacy, trials are ongoing to explore their combinations with temozolomide (TMZ). Here, in order to enhance potency in BRCA1/2-mutant cells, we report on the design of single molecules termed "combi-molecules" capable of not only inhibiting PARP but also damaging DNA like TMZ, which is known to induce a large number of DNA adducts. The majority of these lesions are processed through PARP-dependent base-excision repair machinery. Paradoxically, the least abundant lesion, the O6-methylguanine adduct is the most cytotoxic. Its repair by the O6-methylguanine DNA methyl transferase (MGMT) confers robust resistance to TMZ. Thus, we surmise that a combi-molecule designed to generate the same DNA adducts as TMZ, with an additional ability to block PARP, could induce BRCA1/2 mutant selective potency and a growth inhibitory profile independent of MGMT status.

Methods: The hydrolysis of EG22 and its stabilized form ZSM02 was analyzed by HPLC and fluorescence spectroscopy. Growth inhibitory potency was determined by SRB assay. PARP inhibition was determined by an enzyme assay and DNA damage by the comet assay. Subcellular distribution was visualized by confocal microscopy.

Results: Studies on EG22 showed that: (a) it inflicted anomalously higher levels of DNA damage than TMZ (b) it induced PARP inhibitory potency in the same range as ANI, a known PARP inhibitor (IC50 = 0.10 μM) (c) it showed strong potency in both BRCA1/2 wild type and mutated cells with 6-fold selectivity for the mutants and it was 65-303-fold more potent than TMZ and 4-63-fold than ANI alone and 3-47-fold than their corresponding equimolar combinations and (d) its potency was independent of MGMT expression.

Conclusion: The results in toto suggest that a combi-molecular approach directed at blocking PARP and damaging DNA can lead to single molecules with selective and enhanced potency against BRCA1/2 mutant and with activity independent of MGMT, the major predictive biomarker for resistance to TMZ.

Keywords: 1,2,3-methyltriazene; BRCA1/2 reactivation; Chemoresistance; Combi-targeting; DNA repair; MGMT; PARP inhibitor; Temozolomide.

Fig. 1

Schematic representation of type I and type II combi-molecules. Upon entering the cells, type I combi-molecules are able to bind and inhibit their target as intact molecules. In the cells, the molecule is hydrolyzed to release an inhibitor ‘A’ and a DNA damaging agent ‘B’. Type II combi-molecules enter the cells and are able to inhibit their target and damage DNA without hydrolysis. Inhibition of the target can synergize with the effects of the DNA damaging species

Fig. 2

top: Hydrolysis of TMZ to generate the inactive AIC and the methyl diazonium species; bottom: Hydrolysis of EG22 to regenerate ANI, the naked PARP inhibitor, and the same methyl diazonium species as temozolomide

Fig. 3

Synthesis of ZSM02 from the acetylation of EG22 and its hydrolysis under physiological conditions

Fig. 4

Hydrolysis of EG22 and ZSM02 in serum-containing medium. a The solution of EG22 was kept at 37 °C in the fluorescence reader and an intensity curve automatically generated at the maximum emission wavelength corresponding to ANI (538 nm) (t_1/2 = 9.76 min); b ZSM02 was dissolved in a minimum volume of DMSO and diluted with DMEM supplemented with 10% FBS. The solution was incubated at 37 °C and 100 μL aliquots were analyzed by HPLC as described in Material and Methods. ZSM02 was slowly converted to ANI with t_1/2 greater than 24 h

Fig. 5

Enzymatic assay test for PARP inhibition by measuring the incorporation of biotinylated poly(ADP-ribose) onto histone proteins by the PARP enzyme. This allowed the determination of the IC₅₀ value of our new PARP-DNA combi-molecule, EG22. The Trevigen HT Universal Colorimetric PARP assay kit with histone-coated strip wells was used and dose response curves analyzed with the Graphpad Prism software. The results showed that EG22 was capable of inducing a dose-dependent inhibition of PARP with IC₅₀ = 0.1 μM

Fig. 6

Anomalously strong DNA damaging potential of EG22 after a 2 h drug treatment as compared with temozolomide (TMZ) and ANI + TMZ in the Chinese lung cancer cell lines VC8 and V79. The cells were exposed to the drugs (EG22, TMZ, ANI and ANI + TMZ) for 2 h, and subsequently harvested with trypsin EDTA, centrifuged and resuspended twice in PBS. Comet assay was performed as per Materials and Methods. Comets were visualized at 400X magnification and DNA damage measured as tail moments using Comet Assay IV software

Fig. 7

Dose response curve obtained from growth inhibition with EG22 in a panel of Chinese Hamster Lung cancer cell lines. V79 cells express wild type BRCA2.The VC8 cell line expresses mutant BRCA2, and VC8-BRCA is transfected with the wild type BRCA2 gene. EG22 was significantly more potent against the BCRA2 mutant cell line (p < 0.0001). ZSM02 did not show significant selectivity for VC8 when compared with V79. However, a 3-fold selectivity (p < 0.001) was apparent when compared with its isogenic VC8-BRCA counterpart. *** p < 0.001, ****p < 0.0001, ns. Not Significant

Fig. 8

Proposed pathways for the hydrolysis of EG22 and its dual PARP-DNA targeting property. Solid arrows describe hydrolysis and dotted arrows diffusion. EG22 may diffuse in its intact form through the cell membrane to subsequently hydrolyze in the cytoplasm, release ANI and the methyl diazonium species. ANI may then in turn diffuse into the nucleus and either intercalate into the DNA or inhibit PARP. EG22 may also diffuse in its intact form toward the nucleus, intercalate into DNA prior to being converted to ANI and the methyl diazonium species

Fig. 9

Growth inhibition by EG22 and ZSM02 in a cell panel with varied levels of MGMT. They show similar growth inhibition profile with an increased potency when compared to temozolomide (TMZ), ANI and ANI + TMZ, indicating that their potency is independent of the MGMT status of the cells

Fig. 10

Subcellular distribution of ANI, EG22 and ZSM02 after a 2 h exposure. Following drug treatment, cells were washed with PBS, a drop of DAPI (NucBlue® Live ReadyProbes® Reagent, ThermoFicher Scientific) was added and 3-D images were taken with the appropriate filter. Nuclear localization of the drugs was confirmed by DAPI counterstaining