Direct and Topoisomerase II Mediated DNA Damage by Bis-3-chloropiperidines: The Importance of Being an Earnest G

Abstract

Bis-3-chloropiperidines are a new class of DNA-active compounds capable of alkylating nucleobases and inducing strand cleavage. In this study, we investigated the reactivity of these mustard-based agents with both single- and double-stranded DNA constructs. Polyacrylamide gel electrophoresis (PAGE) and electrospray ionization mass spectrometry (ESI-MS) were used to obtain valuable insight into their mechanism at the molecular level and to investigate their time- and concentration-dependent activity. The results revealed the preferential formation of mono- and bifunctional adducts at nucleophilic guanine sites. In a stepwise fashion, alkylation was followed by depurination and subsequent strand scission at the ensuing apurinic site. We demonstrated that the covalent modifications introduced by this new class of compounds can inhibit the activity of essential DNA-processing proteins, such as topoisomerase IIα, thereby suggesting that bis-3-chloropiperidines may have excellent anticancer potential.

Keywords: DNA alkylation; DNA depurination; anticancer agents; bis-3-chloropiperidines; topoisomerase IIα inhibition.

Figure 1.

A. Chemical structure of representative bis-3-chloropiperidine derivative 1.^[3] B. Chemical structure of the bicyclic aziridinium intermediate, which can be readily attacked by nucleophiles (Nuc). C. The electrophilic bicyclic aziridinium intermediate can be attacked by nucleophilic sites present on either DNA or solvent to give 5- and/or 6-membered ring adducts.

Figure 2.

A. Time- and concentration-dependence of the reaction between bis-3-chloropiperidine 1 and ds-7DG-scr. The duplex construct included a strand carrying a 5’-FAM label to enable visualization and a 7-deaza-2’-deoxyguanosine nucleotide (7DG, chemical structure shown in figure) in position 5. 2 μM aliquots of ds-7DG-scr were treated with either 5 or 50 μM final concentrations of compound 1 at 37 °C in BPE buffer, pH 7.4, and incubated for the time indicated (see Experimental). The reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis (PAA 20 %, 7M urea, TBE 1X). The 50 μM sample incubated for 15 h is labeled “50” and highlighted in red. Arrows indicate the position of cleavage products. C (control) indicates an untreated sample of oligodeoxynucleotide duplex. B. Densitograms used to quantify the intensities of cleavage products. The black line was obtained from the control lane C of panel 2A, while the red line was from the “50” sample, corresponding to the reaction of ds-7DG-scr with 50 μM 1 for 15 h.

Figure 3.

ESI-MS spectrum of reaction mixture obtained by incubating 2 μM of oligodeoxynucleotide ODN1 with 5 μM of bis-3-chloropiperidine 1 at 37 °C for 2 h. Spectra were recorded in 150 mM ammonium acetate (see Experimental Section for conditions). Lower intensity signals near free/bound species consist of typical sodium and ammonium adducts. The inset shows an expanded view of the 4- charge state of the crosslinked ODN1+1_X () and mono-alkylated ODN1+1_OH () adducts.

Figure 4.

Fragments obtained by gas-phase activation of the 3+ charge states of A) ODN1+1_OH and B) ODN1+1_X.

Figure 5.

ESI-MS spectrum of reaction mixture obtained by incubating 2 μM of oligodeoxynucleotide ODN1 with 50 μM of bis-3-chloropiperidine 1 at 37 °C for 2 h. The analysis was performed in 150 mM ammonium acetate (see Experimental for conditions). Lower intensity signals near free/bound species consist of typical sodium and ammonium adducts. The spectrum shows the concentration-dependent reactions induced by compound 1 on ODN1. Only the region containing the 4- charge state is shown. To facilitate the interpretation, we included in the spectrum the graphical representation of identified reaction products (see inset for symbols legend). The symbol corresponds to unmodified oligodeoxynucleotide ODN1; mono-alkylation adduct in which the remaining 3-chloropiperidine function was hydrolyzed to a 3-hydroxyl; crosslinking adduct; base hydrolysis (modified G formally replaced by OH); base elimination (elimination of alkylated G nucleobase).

Scheme 1.

Established mechanism of DNA depurination of N7-alkylguanines residues by bis-3-chloropiperidine 1 and strand cleavage. Guanine residues in DNA (a) can be attacked by reactive bis-3-chloropiperidine 1. The alkylation of the N7 position of guanine places a formal positive charge on the guanine ring system (b), leading to the loss of the alkylguanine. The carbocation intermediate (c) react with the solvent (i.e. water) to form the abasic cyclic acetal (d), which is in equilibrium with small amounts of the ring-opened aldehyde form (e). The loss of the acidic proton adjacent to the carbonyl residue in the aldehydic form of the abasic site can lead to a strand break via elimination of the 3’-phosphate group.^[13]

Figure 6.

ESI-MS spectrum of a sample obtained by incubating 2 μM of the duplex dsDNA with 5 μM of bis-3-chloropiperidine 1 at 37 °C for 2 h. The analysis was performed in 150 mM ammonium acetate (see Experimental for conditions). Lower intensity signals near free/bound species consist of typical sodium and ammonium adducts. For the sake of clarity, the spectrum shows only the region containing the 6- charge state of the duplex and the 3- charge states of its single-stranded components. The symbol corresponds to unmodified single-stranded oligodeoxynucleotide ODN1; single-stranded oligodeoxynucleotide ODN2; double-stranded oligodeoxynucleotide dsDNA; mono-alkylation adduct in which the remaining 3-chloropiperidine function was hydrolyzed to a 3-hydroxyl; crosslinking adduct.

Figure 7.

Effects of bis-3-chloropiperidine 1 on DNA integrity and decatenation by human topoisomerase IIα. Lane B contains kinetoplast DNA (kDNA) used as initial nucleic acid substrate. Lane C contains a mixture of kDNA and topoisomerase IIα (topo IIα) incubated for 1 h at 37°C (see Experimental Section for details). Lane 1, 2, and 3 contain kDNA samples treated respectively with 0.5, 5, and 50 μM of bis-3-chloropiperidine 1, which were incubated for 2 h at 37°C. Lane 1t, 2t, and 3t contain samples with the same compositions of those analyzed in Lane 1–3, which were further incubated with topo IIα for 1 h at 37°C. Lane 4–6 and 4t-6t contain samples analogous to those analyzed in Lane 1–3 and 1t-3t, which were instead reacted for 18 h prior to addition of topo IIα. To facilitate the interpretation, we included arrows to indicate the position of detected decatenated monomers; dashed arrow indicates the position of decatenated monomers migrating more slowly.