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.

Synthesis, Aqueous Reactivity, and Biological Evaluation of Carboxylic Acid Ester-Functionalized Platinum–Acridine Hybrid Anticancer Agents

“…Samples were withdrawn from the incubation mixtures at various time points and quenched with excess thiourea under conditions where the sulfur nucleophile binds to platinum to prevent further reaction with DNA but does not reverse existing DNA adducts. 24 Samples were then analyzed by agarose gel electrophoresis to determine the relative rates of formation of Form 2 of the plasmid. A comparison of the two electrophoretic separations (Figure 6A,B) clearly shows that derivative 9 produces Form 2 at a significantly slower rate than derivative 3´ .…”

The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs

“…289 The most promising second generation derivative was developed by replacing the thiourea donor on Pt-ACRAMTU with an amidine nitrogen. 290,291 This modification accelerated DNA binding, increased cancer cell toxicity by two orders of magnitude (nanomolar IC 50 values), and inhibited tumor growth in vivo. 290,291 Studies in non-small cell lung cancer cells (NCI-H460) suggest that their impressive cytotoxicities can be attributed to rapid intracellular accumulation, DNA adduct formation, and less efficient removal of the DNA adducts.…”

Restriction Inhibition Assay: A Qualitative and Quantitative Method to Screen Sequence Specific DNA Binder from Herbal Plants

“…Moreover, it was much more sensitive in discerning between molecules of comparable structures [11]. This assay was also used to determine the rates of intercalator-driven platination of DNA 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) [12]. Restriction inhibition assay has also been used to measure O…”

“…The assay showed much more sensitivity in discriminating molecules of similar structure such as iso-Dc-81, DC-81 and neothramycin [11]. An assay has been proposed for rapid detection of 0 6 -alkylguanine-DNA alkyltransferase based on restriction endonuclease inhibition in many biological samples [12]. Recently, RIA was used to evaluate the rate of intercalator-driven platination of DNA [13] as well as binding specificity of mitoxantrone which showed more specificity for GC rich sequence [14].…”