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 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am(2) is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k (obs) = 1.4 ± 0.37 × 10(-4) s(-1) (t (1/2) = 83 ± 22 min). 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 ± 2 min), 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 ± 10 min) 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 ± 38 min). 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.

Fig. 1

Structures of platinum–acridines 1–4 with structural modifications in the second-generation derivatives highlighted

Fig. 2

a The cleavage inhibition assay. Incubations of the platinum-treated 40-base-pair duplex (black) with EcoRI and subsequent separation of the mixtures by denaturing polyacrylamide gel electrophoresis (PAGE) results in three detectable products: unmodified (blue) and platinum-modified (green) 18-base-pair fragments, and uncleaved top strand (red). The asterisks denote the ³²P label, and the triangles indicate the sites of endonucleolytic strand scission. b Representative denaturing polyacrylamide gel (12% acrylamide, 8 M urea) for the cleavage inhibition assay performed with compound 2. Lane assignments: C1 control—unmodified probe sequence; C2 control—unmodified, EcoRI-digested sequence; 1–8 reactions with complex 2 quenched after 20, 40 min, 1, 1.5, 2.5, 3.5, 4.5, and 6 h, respectively, followed by EcoRI digestion. The bands are labeled cl. for the 18-base-pair fragment, cl.-Pt for the platinum-containing 18-base-pair fragment, and f.-l. for the full-length fragment

Fig. 3

Proposed mechanism of monofunctional adduct formation

Fig. 4

a Relative integrated band intensities versus reaction time for compound 2. The plotted data are the mean (± the standard deviation) of three individual experiments (gels). Trace assignments: red uncleaved, full-length fragment (f.-l.); blue cleaved 18-base-pair fragment (cl.); green cleaved, platinum-containing 18-base-pair fragment (cl.-Pt); gray calculated sum of “cl.” and “cl.-Pt” band intensities. b Intensities of the “f.-l.” band versus reaction time for compounds 1 (black), 2 (red), 3 (blue), and 4 (green). Each data point is the mean of three individual experiments, with error bars representing ±the standard error of the mean. The solid lines represent calculated best fits to Eq. 2 based on the first-order approximation (see the text)