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

BackgroundMutations 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.MethodsThe 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.ResultsStudies 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.ConclusionThe 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.

15N-, 13C- and 1H-NMR Spectroscopy Characterization and Growth Inhibitory Potency of a Combi-Molecule Synthesized by Acetylation of an Unstable Monoalkyltriazene

“…The synthesis of non-isotopically labeled EG22 ( 8a ) was reported elsewhere [ 14 ]. As shown in Figure 1 , the first evidence of its instability was obtained by monitoring the appearance of a peak corresponding to the shielded proton ortho to the amino group of 4-amino-1,8-naphthalimide (ANI, 7a ) and the disappearance of a deshielded doublet of the aromatic ring of EG22 ( 8a ) in wet DMSO- d 6 at room temperature.…”

“…Indeed, as shown in Figure 7 , using the Sulforhodamine B (SRB) growth inhibitory assay [ 23 ], we confirm that ZSM02 ( 9a ) and EG22 ( 8a ) exhibit similar growth inhibitory profiles. Further work on the mechanism of action of both molecules is reported elsewhere [ 14 ].…”

“…These results in toto suggest that 9a could be a prodrug of 8a. A detailed study on the hydrolysis of 8a and 9a under physiological conditions and on their biological effects as combi-molecules, is reported elsewhere [ 14 ].…”

“…Recently, such a type of molecule 6-(3-methyltriaz-1-en-1-yl)-1 H -benzo[de]isoquinoline-1,3(2 H )-dione, EG22 ( 8a , Scheme 2 ), was designed to behave as an inhibitor of a DNA repair protein termed “poly(adenosine diphosphate ribose) polymerase” (PARP) and a DNA alkylating agent. EG22 ( 8a ) was successfully synthesized and shown to possess dual PARP and DNA targeting properties [ 14 ]. However, its rate of hydrolysis under physiological conditions was too fast and we believed that this could compromise its activity in vivo.…”

Autophagy deficiency downregulates O6methylguanine-DNA methyltransferase and increases chemosensitivity of liver cancer cells

“…MGMT repairs methylated nucleobases in DNA by transferring methyl groups from O 6 -MG of DNA to cysteine residues in the active site of MGMT, resulting in MGMT inactivation and degradation [ 21 ]. Previous studies reported that MGMT was involved in DNA damage caused by the classical alkylating agent temozolomide (TMZ), as TMZ promotes formation of O 6 -methylguanine (O 6 MeG) and breakage of DNA strands [ 22 ]. However, our data showed that the MGMT expression was downregulated in autophagy-deficient liver cancer cells regardless of epirubicin, a non- alkylating agent, suggesting that the MGMT loss was not due to inactivation and degradation caused by repairing the DNA alkyl groups.…”