CHARACTERIZATION OF A CPI-LEXITROPSIN CONJUGATE OLIGONUCLEOTIDE COVALENT COMPLEX BY H-1-NMR AND RESTRAINED MOLECULAR-DYNAMICS SIMULATION

The structural features of the covalent bonding of a novel CPI-lexitro psin conjugate 4 to a model duplex DNA has been examined by high field H-1-NMR analyses and restrained molecular dynamics calculations. Comp ound 4, that was designed for enhanced DNA binding compared with natur al (+)CC-1065, exhibits an exceptional cytotoxic potency against KB hu man nasopharangeal-tumor cells in vitro of IC50 = 0.76 fg/L. Racemic 4 reacted readily with the duplex oligodeoxyribonucleotide d(CGCAATTGCG )(2) to form a single covalent adduct. The latter exhibits a new absor ption band at 396 nm characteristic of the bound drug in addition to t he duplex absorption at 258 nm. H-1-NMR analysis confirms by selective chemical shift changes and NOEs between protons in the drug and in th e duplex that covalent bonding has taken place at A(4). The drug is al igned in a 5'- to 3'-direction at the AATT core in the minor groove re sulting from the selective binding of one enantiomer of 4 and correspo nding to the mode of binding of(-)-CC-1065. The stereochemistry at the site of attachment at the 4a position of the drug is (S), an inferenc e that is corroborated by the restrained molecular dynamics simulation . The latter computations predict average total energies for the (R) a nd (S) drug-DNA adducts of +42 +/- 14.36 and -0.42 +/- 10.34 kcal/mol, respectively, signifying substantially greater stability for the latt er diastereomer. The covalent adduct appears to be quite stable and sh owed no sign of reversibility such as has been observed with other CPI -based agents which may tentatively be attributed to the exceptionally snug fit of all parts of the drug within the minor groove.