Determination of the
DNA-binding reactivity and affinity is an important part of a successful program for the selection of metallodrug candidates. For such assaying, a range of complementary analytical techniques was proposed and tested here using one of few anticancer
metal-based drugs that are currently in clinical trials, indazolium trans-[tetrachloridobis(1H-
indazole)ruthenate(III), and
a DNA oligonucleotide. A high reactivity of the Ru
drug was confirmed in affinity capillary electrophoresis (CE) mode, where adduct formation takes place in situ (i.e., in the capillary filled with an
oligonucleotide-containing
electrolyte). To further characterize the binding kinetics, a
drug-
oligonucleotide mixture was incubated for a different period of time, followed by ultrafiltration separation into two different in molecular weight fractions (>3 and <3 kDa). The time-dependent distribution profiles of the Ru
drug were then assessed by CE-inductively coupled plasma mass spectrometry (ICP-MS), revealing that at least two
DNA adducts exist at equilibrium conditions. Using standalone ICP-MS, dominant equilibrium amount of the bound
ruthenium was found to occur in a fraction of 5-10 kDa, which includes the
oligonucleotide (ca. 6 kDa). Importantly, in all three assays, the
drug was used for the first time in in-vitro studies, not in the intact form but as its active species released from the
transferrin adduct at simulated
cancer cytosolic conditions. This circumstance makes the established analytical platform promising to provide a detailed view on metallodrug targeting, including other possible biomolecules and ex vivo samples.