A new bisintercalating anthracycline with picomolar DNA binding affinity

Abstract

A new bisintercalating anthracycline (WP762) has been designed, in which monomeric units of daunorubicin have been linked through their amino groups on the daunosamine moieties using an m-xylenyl linker. Differential scanning calorimetry and UV melting experiments were used to measure the ultratight binding of WP762 to DNA. The binding constant for the interaction of WP762 with herring sperm DNA was determined to be 7.3 (+/-0.2) x 10(12) M(-1) at 20 degrees C. The large favorable binding free energy of -17.3 kcal mol(-1) was found to result from a large negative enthalpic contribution of -33.8 kcal mol(-1) and an opposing entropic term (-TDeltaS = +16.5 kcal mol(-1)). A comparative molecular modeling study rationalized the increased binding by the m-xylenyl linker of WP762 positioning in the DNA minor groove compared to the p-xylenyl linker found in WP631, the first bis-anthracycline of this type. The cytotoxicity of WP762 was compared to that of other anthracyclines in Jurkat T lymphocytes. These studies, together with an analysis of the cell-cycle traverse in the presence of WP762, suggest that in these cells the new drug is more cytotoxic than the structurally related WP631.

Figure 1

Chemical formulae of daunorubicin and bisantracyclines WP631 and WP762.

Figure 2

UV melting curves for the denaturation of herring sperm DNA (20 μM bp) in the presence of increasing concentrations of WP762. WP762 concentrations were (from left to right): 0, 1, 2, 10, and 20 μM.

Figure 3

Results of DSC experiments. Melting curves are shown for herring sperm DNA at 1 mM (bp) concentration (A) and for an identical DNA solution with saturating amounts (0.16 mol antibiotic/mol bp) of WP762 added (B). The areas under the peaks provide direct estimates of the enthalpy for melting of the DNA duplex (A) and the WP762-DNA complex (B).

Figure 4

Salt dependence of WP762 binding constant at 20°C, compared to daunorubicin and WP631. Data are presented according to the Record’s theory. WP762 (circles), WP631 (diamonds), daunorubicin (squares). Data for daunorubicin and W631 were adapted from. The linear least-squares fit of the data yields a slope (SK) of 1.54 for WP762. From this value, it was estimated that the charge of WP762 is +1.75 at neutral pH.

Figure 5

Molecular models of the average structures of WP631 (A) and WP762 (B), indicating the DNA base pairs and the position of intercalation of each compound.

Figure 6

HINT interaction maps for the interaction of WP631 (A) and WP762 (B) with the 5′-GC|GTAC|GC-3′ base pair sequence of DNA, which displays, visually, the quality and magnitude of the various binding contacts involved in the interaction. The contour surfaces are color-coded by interaction type at a constant map density value of ±150. Blue surfaces represent favorable polar interactions, red surfaces represent unfavorable polar interactions; Green surfaces represent favorable hydrophobic interactions, magenta surfaces represent unfavorable hydrophobic interactions. The interatomic distance between the N3′ ammonium groups on the WP631 and WP762 sugar rings and select atoms of the surrounding base pairs of DNA is also indicated. At right are close-up views of the xylenyl linker region in the average structures of WP631 (C) and WP762 (D), indicating six hydrogen bonds (yellow lines) between the quaternary ammonium and various base pairs in the DNA.

Figure 7

Flow cytometry analyses of Jurkat T cells treated with WP762. Cell cycle distribution was determined at the time intervals indicated. Data indicate the percentage of cells in each phase. (A) Cell cycle distribution of untreated Jurkat T cells. (B) Cell cycle distribution in the presence of 54 nM WP762 (its IC₇₅). Transient changes in G₂/M and sub-G₁ peaks were evident after treatment.

Figure 8

Agarose gel electrophoris analysis of the apoptosis-associated internucleosomal DNA fragmentation in Jurkat T cells treated with 54 nM WP762 (its IC₇₅) for 48 h. For comparison, the effect of 180 nM daunorubicin —its IC₇₅— (Table 2), which produced the typical internucleosomal cleavage in apoptotic cells, is shown as a positive control. Qualitatively, the gel suggested the absence of apoptosis in WP762-treated cells.

Figure 9

Comparison of the binding thermodynamics for several well characterized intercalating compounds: WP631, WP762, Daunorubicin, and Echinomycin.

Scheme 1

Synthesis of WP762.