Molecular analysis of the dual targeting of the epidermal growth factor receptor and the O6-methylguanine-DNA methyltransferase with a double arm hybrid molecule

Abstract

Disordered expression of the epidermal growth factor receptor (EGFR) has been associated with induction of DNA repair genes (e.g. XRCC1, ERCC1) and resistance to radiation and genotoxic drugs. However, our previous work showed that EGFR inhibition did not affect O6-methylguanine-DNA methyltransferase (MGMT)-mediated resistance. In order to block uncoupled events associated with EGFR and MGMT, we designed MR30, a single molecule termed “combi-molecule” that contains a quinazoline arm targeted to EGFR and an O6-benzylguanine (O6-BG) moiety to block MGMT. Molecular analysis of the mechanism of action of its two arms showed that: (a) it could block EGFR phosphorylation, (b) down-regulate the RAF-MAPK and the PI3K-AKT pathways, and (c) covalently modify MGMT through S-benzylation, as confirmed by MALDI analysis of a direct binding assay with isolated MGMT, (d) it induced a dose-dependent down-regulation of MGMT in lung and melanoma cells. The pleiotropic mechanism of action of MR30 culminated into strong growth inhibition (IC50: 0.018-6.02 μM), with superior activity when compared with an equimolar combination of gefitinib (a clinical EGFR inhibitor) and O6-BG (a known MGMT inhibitor). Pulse exposure experiments were required to attenuate the contribution of EGFR inhibition to the strong potency of MR30, thereby allowing to achieve the dose level required to sensitize cells to temozolomide (TMZ). Indeed, MR30 significantly sensitized EGFR-MGMT co-expressing cells to TMZ (p<0.05-0.0001). The results in toto suggest that MR30 is the first prototype of agents that may be used against tumours addicted to EGFR and to sensitize resistant tumours co-expressing EGFR and MGMT to TMZ.

Acridine–O6-benzylguanine hybrids: Synthesis, DNA binding, MGMT inhibition and antiproliferative activity

“…In drug combination experiments, compounds were added to cell cultures 2 h prior to addition of TMZ, following by incubation for 94 h in the presence of both drugs, which is a typical procedure for the assessment of MGMT inhibitors [11][12][13]38,56,60,61]. Bliss Independence model was used to analyze the combination effects of the drugs.…”

“…By definition, hybrid drugs contain two different pharmacophore units that can amplify or modulate each other's activity, displaying a dual activity inside a cell. With regard to MGMT inhibitors, the BG moiety was exploited in the design of a hybrid drug MR30 by linking to a quinazoline derivative targeting the epidermal growth factor receptor (EGFR) in A549 cell lines [11]. In parallel, BG has been linked to DNA-alkylating nitrosourea derivatives; the resulting hybrid drugs showed markedly increased cytotoxicity with regard to clinically used nitrosoureas or their combinations with BG in several human glioma cells lines such as SF126, SF763 or SF767 [12,13].…”

Quantitative Analysis of the Potency of Equimolar Two-Drug Combinations and Combi-Molecules Involving Kinase Inhibitors In Vitro: The Concept of Balanced Targeting

“…Thus, in recent years, strategies designed to overcome resistance mediated by compensatory signaling have involved the use of a multi-targeted approach [4,5]. Within this context, over the past decade, we developed a novel approach termed "combi-targeting" that sought to design agents designated as "combi-molecules" capable of inducing tandem blockade of two divergent biological targets (e.g., EGFR, PARP, MEK, and DNA) [6][7][8][9][10][11][12][13][14][15][16]. Further work on the concept led to the synthesis of molecules rationally designed to target two oncogenic tyrosine kinases involved in adverse signaling [17,18].…”

Design and Synthesis of a Trifunctional Molecular System “Programmed” to Block Epidermal Growth Factor Receptor Tyrosine Kinase, Induce High Levels of DNA Damage, and Inhibit the DNA Repair Enzyme (Poly(ADP-ribose) Polymerase) in Prostate Cancer Cells

“…Recently, with the purpose of selectively sensitizing resistant O6-methylguanine DNA methyl transferase (MGMT)-expressing cells co-expressing EGFR to the DNA alkylating drug temozolomide, we designed MR30 , a binary type II combi-molecule, and demonstrated its ability to block EGFR and deplete MGMT . However, this system was not able to induce a triple-targeting functions as an intact combi-molecule as it required an exogenous source of DNA damage to be active against resistant cells.…”

“…15 Recently, with the purpose of selectively sensitizing resistant O6-methylguanine DNA methyl transferase (MGMT)-expressing cells co-expressing EGFR to the DNA alkylating drug temozolomide, we designed MR30, a binary type II combi-molecule, and demonstrated its ability to block EGFR and deplete MGMT. 16 However, this system was not able to induce a triple-targeting functions as an intact combi-molecule as it required an exogenous source of DNA damage to be active against resistant cells. To circumvent this problem, here, we advanced our concept toward the design of the first ternary system (I 1 −I 2 −I 3 ) to contain an EGFR-targeting leading edge (I 1 ), a masked DNA damaging function (I 2 ), and more importantly, a DNA repair inhibitory (PARP) moiety (I 3 ), which is added to block the repair of the types of DNA lesions that the system is designed to induce (Figure 2).…”