Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay

Abstract

A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum-acridine agents represented by the formula [Pt(am(2))LCl](NO(3))(2), where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5'-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am(2)isethane-1,2-diamine, LisACRAMTU) reacts with a first-order rate constant of k (obs)=1.4±0.37×10(-4)s(-1) (t (1/2)=83±22min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k (obs)=5.7±0.58×10(-4)s(-1), t (1/2)=21±2min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k (obs)=2.1±0.40×10(-4)s(-1), t (1/2)=55±10min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k (obs)=1.1±0.40×10(-4)s(-1), t (1/2)=104±38min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine-purine cross-links are discussed.