Subcellular Distribution of a Fluorescence-Labeled Combi-Molecule Designed to Block Epidermal Growth Factor Receptor Tyrosine Kinase and Damage DNA with a Green Fluorescent Species

Abstract

To monitor the subcellular distribution of mixed epidermal growth factor (EGF) receptor (EGFR)-DNA targeting drugs termed combi-molecules, we designed AL237, a fluorescent prototype, to degrade into a green fluorescent DNA damaging species and FD105, a blue fluorescent EGFR inhibitor. Here we showed that AL237 damaged DNA in the 12.5 to 50 μmol/L range. Despite its size, it blocked EGFR phosphorylation in an enzyme assay (IC 50 = 0.27 μmol/L) and in MDA-MB468 breast cancer cells in the same concentration range as for DNA damage. This translated into inhibition of extracellular signal-regulated kinase 1/2 or BAD phosphorylation and downregulation of DNA repair proteins (XRCC1, ERCC1). Having shown that AL237 was a balanced EGFR-DNA targeting molecule, it was used as an imaging probe to show that (a) green and blue colors were primarily colocalized in the perinuclear and partially in the nucleus in EGFR-or ErbB2-expressing cells, (b) the blue fluorescence associated with FD105, but not the green, was colocalized with anti-EGFR redlabeled antibody, (c) the green fluorescence of nuclei was significantly more intense in NIH 3T3 cells expressing EGFR or ErbB2 than in their wild-type counterparts (P < 0.05). Similarly, the growth inhibitory potency of AL237 was selectively stronger in the transfectants. In summary, the results suggest that AL237 diffuses into the cells and localizes abundantly in the perinuclear region and partially in the nucleus where it degrades into EGFR and DNA targeting species. This bystander-like effect translates into high levels of DNA damage in the nucleus. Sufficient quinazoline levels are released in the cells to block EGF-induced activation of downstream signaling. Mol Cancer Ther; 9(4); 869-82. ©2010 AACR.

The Combi-Targeting Concept: Selective Targeting of the Epidermal Growth Factor Receptor- and Her2-Expressing Cancer Cells by the Complex Combi-Molecule RB24

“…We recently made the same observation with AL237, a fluorescencelabeled probe that allowed us to localize the DNA-alkylating moiety by fluorescence microscopy. It is more important that by using red-labeled EGFR antibody, we demonstrated that the released FD105, the chloro analog of RB10, was colocalized with EGFR (Todorova et al, 2010). It also should be noted that, in addition to our recent imaging work, there is now ample evidence of the perinuclear and even nuclear translocation of EGFR (Lo et al, 2006;Dittmann et al, 2009).…”

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

“…Through the mitogen-activated protein kinase (MAPK) pathway, EGFR activation leads to expression of DNA repair genes [ 11 ] and through the phosphatidylinositol-3 kinase (PI3K) pathway, it exerts an antiapoptotic effect [ 12 ]. Based on the premise that activation of EGFR leads to induction of DNA repair proteins such as X-ray repair cross-complementing protein 1 (XRCC1) and excision repair cross-complementation group 1 (ERCC1), our group embarked onto the design of drugs termed “combi-molecules” that can induce a tandem targeting of EGFR and DNA and demonstrated that indeed the latter dual targeted molecules could down-regulate XRCC1 and ERCC1 through their EGFR inhibitory arm and inducing high levels of DNA strand breaks through their DNA alkylating arm [ 11 ]. We showed that the dual targeted approach termed “combi-targeting” translated into massive cell death by apoptosis.…”

Target Modulation by a Kinase Inhibitor Engineered to Induce a Tandem Blockade of the Epidermal Growth Factor Receptor (EGFR) and c-Src: The Concept of Type III Combi-Targeting

“…In the past, in the context of a novel multi-targeted approach termed “combi-targeting”, we designed inhibitors termed “combi-molecules” that can block targets as divergent as tyrosine kinase receptors and genomic DNA. We demonstrated their ability to kill tumour cells by blocking receptor phosphorylation, damaging DNA and down-regulating DNA repair proteins [ 4 , 5 ]. We classified such molecules as type I (i.e., those that require hydrolysis to fully exhibit their dual potency) and type II (i.e., those that could induce DNA damage and a tandem blockade of receptor mediated signaling without requirement for hydrolysis).…”