title: Intracellular protein binding patterns of the anticancer ruthenium drugs KP1019 and KP1339.
abstract: 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.
authors.affiliation: Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Heffeter
authors.forename: Katharina
authors.initials: K
authors.lastname: Böck
authors.forename: Bihter
authors.initials: B
authors.lastname: Atil
authors.forename: Mir Ali
authors.initials: MA
authors.lastname: Reza Hoda
authors.forename: Wilfried
authors.initials: W
authors.lastname: Körner
authors.forename: Caroline
authors.initials: C
authors.lastname: Bartel
authors.forename: Ute
authors.initials: U
authors.lastname: Jungwirth
authors.forename: Bernhard K
authors.initials: BK
authors.lastname: Keppler
authors.forename: Michael
authors.initials: M
authors.lastname: Micksche
authors.forename: Walter
authors.initials: W
authors.lastname: Berger
authors.forename: Gunda
authors.initials: G
authors.lastname: Koellensperger
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D049109:Cell Proliferation / Q000187:drug effects; D003600:Cytosol / Q000737:chemistry; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D007191:Indazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013058:Mass Spectrometry; D008970:Molecular Weight; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D017975:Ruthenium Compounds; D013329:Structure-Activity Relationship; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D007191:Indazoles; C551585:KP 1339; D009942:Organometallic Compounds; D011506:Proteins; D017975:Ruthenium Compounds; C499292:indazolium trans-(tetrachlorobis(1H-indazole)ruthenate (III))