Unprecedented Monofunctional Metalation of Adenine Nucleobase in Guanine- and Thymine-Containing Dinucleotide Sequences by a Cytotoxic Platinum−Acridine Hybrid Agent

Abstract

We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO(3))(2) (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy ((1)H, (195)Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)](3+) (P1, major) and [Pt(en)(dGuo)(2)](2+) (P2, minor). The reaction, which proceeds significantly slower (half-life 11-12 h at 37 degrees C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in approximately 15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)](3+) (A1, approximately 80%), [Pt(en)(ACRAMTU)[d(GpA)]](2+) (A2, approximately 12%), and [Pt(en)(ACRAMTU)[d(TpA)]](2+) (A3, approximately 8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.

Characterization of the bisintercalative DNA binding mode of a bifunctional platinum-acridine agent

“…PT-ACRAMTU ([PtCl(en)(ACRAMTU)](NO 3 ) 2 , en = ethylenediamine; Figure 1) and its derivatives show promising activity in vitro in several solid tumors, including ovarian and lung cancer cell lines (15–18). While the cellular biology of the parent conjugate is still under investigation, we proposed the unprecedented targeting of adenine residues at 5′-TA sites in the minor groove of DNA by this dual metalating-intercalating agent (19,20) may play an important role in its mechanism of action (21–23). Following our interest in small-molecule modulators of transcription, we have now begun to extend our studies to reversible bifunctional intercalators derived from PT-ACRAMTU.…”

Potential adenine and minor groove binding platinum complexes

“…Generally, the modified platinum drugs form an array of covalent adducts similar to that formed by cisplatin. However, Bierbach and co-workers have reported the first example of monofunctional adenine adducts being formed by a divalent platinum complex, [PtCl(en)(ACRAMTU-S] (ACRAMTU ¼ 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea), with double-stranded DNA [37]. Furthermore, the authors concluded that the platinum-acridine conjugate targets adenine residues in the DNA minor groove, with covalent binding possibly at the N3 site [37].…”

“…However, Bierbach and co-workers have reported the first example of monofunctional adenine adducts being formed by a divalent platinum complex, [PtCl(en)(ACRAMTU-S] (ACRAMTU ¼ 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea), with double-stranded DNA [37]. Furthermore, the authors concluded that the platinum-acridine conjugate targets adenine residues in the DNA minor groove, with covalent binding possibly at the N3 site [37]. Alternatively, given that platinum complexes predominantly form covalent adducts in the DNA major groove, it may be possible to utilise inert octahedral complexes that specifically bind in the DNA major groove in the linking ligand.…”

Guanine binding of a cytotoxic platinum–acridin-9-ylthiourea conjugate monitored by 1-D 1H and 2-D [1H,15N] NMR spectroscopy: Hydrolysis is not the rate-determining step

“…1; [PtCl(en)(ACRAM-TUS)](NO 3 ) 2 ; ACRAMTU = 1-[2-(acridin-9-ylamino)-ethyl]-1,3-dimethylthiourea, en = ethane-1,2-diamine) was designed to produce DNA adducts different from those formed by the clinical platinum agents [5]. Using enzymatic and acidic digestion assays we have demonstrated previously that complex 1 forms monofunctional adducts with guanine (G) ($80%) and adenine (A) ($20%) in native DNA [6]. In all of the adducts identified, the chloro leaving group has been replaced with nucleobase nitrogen (G-N7, A-N7, A-N3, or A-N1), while the sulfur-bound intercalator remains attached to the metal, rendering ACRAMTU a typical non-leaving group [7,8].…”

“…A half-life of adduct formation of 7.5 h was determined from timedependent NMR spectra when 1 mM Pt was incubated with 3 mM of dG at 37°C [10]. In contrast, reactions of conjugate 1 with calf thymus DNA proceed significantly faster and are virtually complete after 12 h [6]. Thus, we speculated that platination of nucleobase nitrogen in double-stranded DNA from a preintercalated state might be kinetically favored over simple ligand substitution in the mononucleoside model system.…”