Karla Pelivan

Impact of Stepwise NH2-Methylation of Triapine on the Physicochemical Properties, Anticancer Activity, and Resistance Circumvention

One of the most promising classes of iron chelators are α-N-heterocyclic thiosemicarbazones with Triapine as the most prominent representative. In several clinical trials Triapine showed anticancer activity against hematological diseases, however, studies on solid tumors failed due to widely unknown reasons. Some years ago, it was recognized that “terminal dimethylation” of thiosemicarbazones can lead to a more than 100-fold increased activity, probably due to interactions with cellular copper depots. To better understand the structural requirements for the switch to nanomolar cytotoxicity, we systematically synthesized all eight possible N-methylated derivatives of Triapine and investigated their potential against Triapine-sensitive as well as -resistant cell lines. While only the “completely” methylated compound exerted nanomolar activity, the data revealed that all compounds with at least one N-dimethylation were not affected by acquired Triapine resistance. In addition, these compounds were highly synergistic with copper treatment accompanied by induction of reactive oxygen species and massive necrotic cell death.

Triapine-mediated ABCB1 induction via PKC induces widespread therapy unresponsiveness but is not underlying acquired triapine resistance

Although triapine is promising for treatment of advanced leukemia, it failed against solid tumors due to widely unknown reasons. To address this issue, a new triapine-resistant cell line (SW480/tria) was generated by drug selection and investigated in this study. Notably, SW480/tria cells displayed broad cross-resistance against several known ABCB1 substrates due to high ABCB1 levels (induced by promoter hypomethylation). However, ABCB1 inhibition did not re-sensitize SW480/tria cells to triapine and subsequent analysis revealed that triapine is only a weak ABCB1 substrate without significant interaction with the ABCB1 transport function. Interestingly, in chemo-naive, parental SW480 cells short-time (24 h) treatment with triapine stimulated ABCB1 expression. These effects were based on activation of protein kinase C (PKC), a known response to cellular stress. In accordance, SW480/tria cells were characterized by elevated levels of PKC. Together, this led to the conclusion that increased ABCB1 expression is not the major mechanism of triapine resistance in SW480/tria cells. In contrast, increased ABCB1 expression was found to be a consequence of triapine stress-induced PKC activation. These data are especially of importance when considering the choice of chemotherapeutics for combination with triapine.

Differences in protein binding and excretion of Triapine and its Fe(III) complex

Triapine has been investigated as anticancer drug in multiple clinical phase I/II trials. Although promising anti-leukemic activity was observed, Triapine was ineffective against solid tumors. The reasons are currently widely unknown. The biological activity of Triapine is strongly connected to its iron complex (Fe-Triapine) which is pharmacologically not investigated. Here, novel analytical tools for Triapine and Fe-Triapine were developed and applied for cell extracts and body fluids of treated mice. Triapine and its iron complex showed a completely different behavior: for Triapine, low protein binding was observed in contrast to fast protein adduct formation of Fe-Triapine. Notably, both drugs were rapidly cleared from the body (serum half-life time <1h). Remarkably, in contrast to Triapine, where (in accordance to clinical data) basically no renal excretion was found, the iron complex was effectively excreted via urine. Moreover, no Fe-Triapine was detected in serum or cytosolic extracts after Triapine treatment. Taken together, our study will help to further understand the biological behavior of Triapine and its Fe-complex and allow the development of novel thiosemicarbazones with pronounced activity against solid tumor types.

EGFR-targeting peptide-coupled platinum(IV) complexes

The high mortality rate of lung cancer patients and the frequent occurrence of side effects during cancer therapy demonstrate the need for more selective and targeted drugs. An important and well-established target for lung cancer treatment is the occasionally mutated epidermal growth factor receptor (EGFR). As platinum(II) drugs are still the most important therapeutics against lung cancer, we synthesized in this study the first platinum(IV) complexes coupled to the EGFR-targeting peptide LARLLT (and the shuffled RTALLL as reference). Notably, HPLC–MS measurements revealed two different peaks with the same molecular mass, which turned out to be a transcyclization reaction in the linker between maleimide and the coupled cysteine moiety. With regard to the EGFR specificity, subsequent biological investigations (3-day viability, 14-day clonogenic assays and platinum uptake) on four different cell lines with different verified EGFR expression levels were performed. Unexpectedly, the results showed neither an enhanced activity nor an EGFR expression-dependent uptake of our new compounds. Consequently, fluorophore-coupled peptides were synthesized to re-evaluate the targeting ability of LARLLT itself. However, also with these molecules, flow cytometry measurements showed no correlation of drug uptake with the EGFR expression levels. Taken together, we successfully synthesized the first platinum(IV) complexes coupled to an EGFR-targeting peptide; however, the biological investigations revealed that LARLLT is not an appropriate peptide for enhancing the specific uptake of small-molecule drugs into EGFR-overexpressing cancer cells.

Synthesis and biological evaluation of biotin-conjugated anticancer thiosemicarbazones and their iron(III) and copper(II) complexes

Triapine, the most prominent anticancer drug candidate from the substance class of thiosemicarbazones, was investigated in >30 clinical phase I and II studies. However, the results were rather disappointing against solid tumors, which can be explained (at least partially) due to inefficient delivery to the tumor site. Hence, we synthesized the first biotin-functionalized thiosemicarbazone derivatives in order to increase tumor specificity and accumulation. Additionally, for Triapine and one biotin conjugate the iron(III) and copper(II) complexes were prepared. Subsequently, the novel compounds were biologically evaluated on a cell line panel with different biotin uptake. The metal-free biotin-conjugated ligands showed comparable activity to the reference compound Triapine. However, astonishingly, the metal complexes of the biotinylated derivative showed strikingly decreased anticancer activity. To further analyze possible differences between the metal complexes, detailed physico- and electrochemical experiments were performed. However, neither lipophilicity or complex solution stability, nor the reduction potential or behavior in the presence of biologically relevant reducing agents showed strong variations between the biotinylated and non-biotinylated derivatives (only some differences in the reduction kinetics were observed). Nonetheless, the metal-free biotin-conjugate of Triapine revealed distinct activity in a colon cancer mouse model upon oral application comparable to Triapine. Therefore, this type of biotin-conjugated thiosemicarbazone is of interest for further synthetic strategies and biological studies.

Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187

HIGHLIGHTSProton dissociation constants (pKa) of epidermal growth factor receptor inhibitors.Binding affinity to HSA studied by fluorometry and molecular docking calculations.Model calculations for HSA binding at physiological blood concentrations.Binding data firstly provided for osimertinib and investigational drug KP2187. ABSTRACT 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.

Comparison of metabolic pathways of different α-N-heterocyclic thiosemicarbazones

AbstractClinical failure of novel drugs is often related to their rapid metabolism and excretion. This highlights the importance of elucidation of their pharmacokinetic profile already at the preclinical stage of drug development. Triapine, the most prominent representative of α-N-heterocyclic thiosemicarbazones, was investigated in more than 30 clinical phase I/II trials, but the results against solid tumors were disappointing. Recent investigations from our group suggested that this is, at least partially, based on the fast metabolism and excretion. In order to establish more detailed structure/activity/metabolism relationships, herein a panel of 10 different Triapine derivatives was investigated for their metabolic pathways. From the biological point of view, the panel consists of terminally dimethylated thiosemicarbazones with nanomolar IC50 values, derivatives with micromolar cytotoxicities comparable to Triapine and a completely inactive representative. To study the oxidative metabolism, a purely instrumental approach based on electrochemistry/mass spectrometry was applied and the results were compared to the data obtained from microsomal incubations. Overall, the investigated thiosemicarbazones underwent the phase I metabolic reactions dehydrogenation, hydroxylation, oxidative desulfuration (to semicarbazone and amidrazone) and demethylation. Notably, dehydrogenation resulted in a ring-closure reaction with formation of thiadiazoles. Although strong differences between the metabolic pathways of the different thiosemicarbazones were observed, they could not be directly correlated to their cytotoxicities. Finally, the metabolic pathways for the most cytotoxic compound were elucidated also in tissues collected from drug-treated mice, confirming the data obtained by electrochemical oxidation and microsomes. In addition, the in vivo experiments revealed a very fast metabolism and excretion of the compound. Graphical abstractStructure/activity/metabolisation relationships for 10 anticancer thiosemicarbazones were established using electrochemical oxidation coupled to mass spectrometry (EC-MS) and human liver microsomes analyzed by LC-MS

Abstract 5461: Triapine-mediated ABCB1 induction via PKC induces widespread therapy unresponsiveness but is not underlying acquired triapine resistance

Due to their enhanced proliferation rate, tumor cells are highly susceptible for ribonucleotide pool disruption. Therefore, several thiosembicarbazone-based ribonucleotide reductase inhibitors have been developed, out of which Triapine is the most promising candidate. Triapine is currently tested in clinical phase I and II studies and shows promising effects in haematological diseases. Unfortunately, triapine is rather ineffective in solid cancer types. However, the mechanism underlying this failure is yet not fully understood. One possible theory could be development of rapid acquired resistance against the chemotherapeutic drug. To investigate this issue we generated a triapine-resistant cell line (SW480/tria) by stepwise selection of human colon carcinoma SW480 cells. SW480/tria cells displayed a broad cross-resistance especially against several well-known ABCB1 substrates (e.g. vincristine). In accordance, strong ABCB1 expression was detected in SW480 Tria cells. The induction of ABCB1 was not based on gene amplification but on hypomethylation of the ABCB1 promoter. As a next step, rhodamine-123 and ATPase assays were performed to investigate if triapine does interact with the ABCB1 transport function. However, no ABCB1 inhibitory potential of triapine could be found. Further on, combined treatment in SW480/tria cells with triapine and the known ABCB1 inhibitors cyclosporine A (CSA) and verapamil did not restore triapine sensitivity. In addition, the intracellular triapine levels were comparable between parental and triapine-selected SW480 cell lines. Moreover, increased ABCB1 expression was found to be a consequence of triapine stress-induced PKC activation. This suggests that the strong ABCB1 expression of SW480/tria cells might rather be a consequence of triapine-induced cell stress than a major driver for specific triapine resistance. Taken together our data reveal that, although triapine is only a weak substrate for ABCB1, strong ABCB1 expression is induced by short- and long-term triapine exposure resulting in distinct cross resistance against other anticancer drugs. This definitely has to be considered in the selection of combination schemes and in second line therapy following triapine failure. Citation Format: Walter Miklos, Karla Pelivan, Christian Kowol, Rita Dornetshuber-Fleiss, Melanie Spitzwieser, Margit Cichna-Markl, Gunda Kollensperger, Bernhard Keppler, Walter Berger, Petra Heffeter. Triapine-mediated ABCB1 induction via PKC induces widespread therapy unresponsiveness but is not underlying acquired triapine resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5461. doi:10.1158/1538-7445.AM2015-5461