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Binding interactions between human serum albumin (HSA) and four approved epidermal growth factor receptor (EGFR) inhibitors gefitinib (GEF), erlotinib (ERL), afatinib (AFA), osimertinib (OSI), as well as the experimental drug KP2187, were investigated by means of spectrofluorometric and molecular modelling methods. Steady-state and time resolved spectrofluorometric techniques were carried out, including direct quenching of protein fluorescence and site marker displacement measurements. Proton dissociation processes and solvent dependent fluorescence properties were investigated as well. The EGFR inhibitors were predominantly presented in their single protonated form (HL + ) at physiological pH except ERL, which is charge-neutral. Significant solvent dependent fluorescence properties were found for GEF, ERL and KP2187, namely their emission spectra show strong dependence on the polarity and the hydrogen bonding ability of the solvents. The inhibitors proved to be bound at site I of HSA (in subdomain IIA) in a weak-to-moderate fashion (logK' 3.9-4.9) using spectrofluorometry. OSI (logK' 4.3) and KP2187 can additionally bind in site II (in subdomain IIIA), while GEF, ERL and AFA clearly show no interaction here. Docking methods qualitatively confirmed binding site preferences of compounds GEF and KP2187, and indicated that they probably bind to HSA in their neutral forms. Binding constants calculated on the basis of the various experimental data indicate a weak-to-moderate binding on HSA, only OSI exhibits somewhat higher affinity towards this protein. However, model calculations performed at physiological blood concentrations of HSA resulted in high (ca. 90%) bound fractions for the inhibitors, highlighting the importance of plasma protein binding., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Triapine is an α-N-heterocyclic thiosemicarbazone with promising anticancer activity against hematologic malignancies but widely ineffective against solid tumor types in clinical trials. The anticancer activity of thiosemicarbazones can be dramatically increased by terminal dimethylation. KP1089 is a gallium compound containing two terminal dimethylated thiosemicarbazone ligands. To gain insights on the vulnerability of this highly active terminal dimethylated thiosemicarbazone to drug resistance mechanisms, a new cell model with acquired resistance against the lead compound KP1089 was established. Subsequent genomic analyses (arrayCGH and FISH) revealed amplification of the ABCC1 gene on double minute chromosomal DNA in KP1089-resistant cells as well as overexpression of ABCC1 and ABCG2 on the protein level. KP1089 was further confirmed as a substrate of ABCC1 and ABCG2 but not of ABCB1 using a panel of ABC transporter-overexpressing cell models as well as ABC transporter inhibitors. Moreover, glutathione depletion strongly enhanced KP1089 activity, although no glutathione conjugate formation by glutathione-S-transferase was observed. Thus, a co-transport of KP1089 together with glutathione is suggested. Finally, a panel of thiosemicarbazone derivatives was tested on the new KP1089-resistant cell line. Notably, KP1089-resistant cells were not cross-resistant against thiosemicarbazones lacking terminal dimethylation (e.g. Triapine) which are less active than KP1089. This suggests that terminal dimethylation of thiosemicarbazones - linked with distinctly enhanced anticancer activity - leads to altered resistance profiles compared to classical thiosemicarbazones making this compound class of interest for further (pre)clinical evaluation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Microemulsion electrokinetic chromatography (MEEKC) offers a valuable tool for the rapid and highly productive determination of lipophilicity for metal-based anticancer agents. In this investigation, the MEEKC technique was applied for estimation of n-octanol-water partition coefficient (logP(oct)) of a series of antiproliferative complexes of gallium(III) and iron(III) with (4)N-substituted α-N-heterocyclic thiosemicarbazones. Analysis of relationships between the experimental logP(oct) and the retention factors of compounds showed their satisfactory consistency in the case of single metal sets, as well as for both metals. Since none of available calculation programs allows for evaluating the contribution of central metal ion into logP(oct) (i.e. ΔlogP(oct)) of complexes of different metals, this parameter was measured experimentally, by the standard 'shake-flask' method. Extension of the logP(oct) programs by adding ΔlogP(oct) data resulted in good lipophilicity predictions for the complexes of gallium(III) and iron(III) included in one regression set. Comparison of metal-thiosemicarbazonates under examination in terms of logP(oct) vs. antiproliferative activities (i.e. 50% inhibitory concentration in cancer cells) provided evidence that their cytotoxic potency is associated with the ability to cross the lipid bilayer of the cell-membrane via passive diffusion., (Copyright © 2011 Elsevier B.V. All rights reserved.)