The
ruthenium compound
KP1019 has demonstrated promising anticancer activity in a pilot clinical trial. This study aims to evaluate the intracellular uptake/binding patterns of
KP1019 and its
sodium salt KP1339, which is currently in a phase I-IIa study. Although
KP1339 tended to be moderately less cytotoxic than
KP1019, IC(50) values in several
cancer cell models revealed significant correlation of the cytotoxicity profiles, suggesting similar targets for the two drugs. Accordingly, both drugs activated apoptosis, indicated by
caspase activation via comparable pathways.
Drug uptake determined by inductively coupled plasma mass spectrometry (ICP-MS) was completed after 1 h, corresponding to full cytotoxicity as early as after 3 h of
drug exposure. Surprisingly, the total cellular
drug uptake did not correlate with cytotoxicity. However, distinct differences in intracellular distribution patterns suggested that the major targets for the two
ruthenium drugs are cytosolic rather than nuclear. Consequently,
drug-protein binding in cytosolic fractions of
drug-treated cells was analyzed by native size-exclusion chromatography (SEC) coupled online with ICP-MS.
Ruthenium-protein binding of KP1019- and KP1339-treated cells distinctly differed from the
platinum binding pattern observed after
cisplatin treatment. An adapted SEC-SEC-ICP-MS system identified large
protein complexes/aggregates above 700 kDa as initial major binding partners in the cytosol, followed by
ruthenium redistribution to the soluble
protein weight fraction below 40 kDa. Taken together, our data indicate that
KP1019 and
KP1339 rapidly enter
tumor cells, followed by binding to larger
protein complexes/organelles. The different protein binding patterns as compared with those for
cisplatin suggest specific
protein targets and consequently a unique mode of action for the
ruthenium drugs investigated.