DNA interaction of the tyrosine protein kinase inhibitor PD153035 and its N-methyl analogue

The brominated anilinoquinazoline derivative BD153035 exhibits a very high affinity and selectivity for the epidermal growth factor receptor tyrosine kinase (EGF-R TK) and shows a remarkable cytotoxicity against several types of tumor cell lines. In contrast, its N-methyl derivative, designated EBE- A22, has no effect on EGF-R TK but maintains a high cytotoxic profile. The present study was pet-formed to explore the possibility that PB153035 and i ts N-methyl analogue might interact with double-stranded DNA, which is a pr imary target for many conventional antitumor agents. We studied the strengt h and mode of binding to DNA of PD 153035 and EBE-A22 by means of absorptio n, fluorescence, and circular and linear dichroism as well as by a relaxati on assay using human DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to show that both drugs bind to DN A and behave as typical intercalating agents. In particular, EBE-A22 unwind s supercoiled plasmid, stabilizes duplex DNA against heat denaturation, and produces negative CD and ELD signals, as expected for an intercalating age nt. Extensive DNase I footprinting experiments performed with a large range of DNA substrates show that EBE-A22, but not PD153035, interacts preferent ially with CC-rich sequences and discriminates against homooligomeric runs of A and T which are often cut more readily by the enzyme in the presence o f the drug compared to the control. Altogether, the results provide the fir st experimental evidence that DNA is a target of anilinoquinazoline derivat ives and suggest that this N-methylated ring system is a valid candidate fo r the development of DNA-targeted cytotoxic compounds. The possible relevan ce of selective DNA binding to activity is considered. The unexpected GC-se lective binding properties of EBE-A22 entreat further exploration into the use of N-methylanilinoquinazoline derivatives as tools for designing sequen ce-specific DNA binding ligands.