New thiosemicarbazone-based anticancer drugs

Chemotherapy and therapy with small targeted biologicals are two major strategies to combat human cancer at the disseminated stage. Due to high iron dependence, cancer cells are known for their sensitivity against iron deprivation. In order to target this Archilles heel of malignant cells, several iron chelators have been developed. Iron chelation by thiosemicarbazones has been reported repeatedly to target the iron centre of the ribonucleotide reductase, an enzyme essential for dNTP pool maintenance and DNA synthesis. Additionally, other mechanisms such as topoisomerase II inhibition but also yet undefined targets might be involved in thiosemicarbazone anticancer activity. Most promising candidate among the thiosemicarbazone-based drugs is 3-aminopyridine-2- carboxaldehyde thiosemicarbazone (Triapine) as lead compound currently under clinical evaluation. However, several clinical phase II studies revealed that Triapine, despite promising activity in advanced hematologic malignancies, is ineffective against several solid tumors. The reasons for this observation are currently unknown and might be based on inappropriate drug delivery into the solid tumor nodules. Since several years researcher groups at the Institute for Cancer Research/Department of Medicine I (Medical University Vienna) and the Institute for Inorganic Chemistry (University Vienna) synergistically cooperate with the aim to develop novel anticancer (metal) drugs. These activities, also supported be the FWF (e.g. L12, L568, P18123, P20897, P20683), recently has culminated in the establishment of an inter-university cooperation platform on Translational Cancer Therapy Research. Main focus of this proposed project is the development of novel derivatives and metal complexes of Triapine with enhanced tumour selectivity/activity. Additionally, we aim on dissecting the molecular modes of action underlying the anticancer activity of these drugs. To this end, several rational strategies are followed: 1) introduction of several new methyl groups to modulate general cytotoxicity, 2) coordination of Triapine and derivatives to copper to enhance hypoxia-targeting, and 3) linkage of thiosemicarbazones to several functional groups to increase drug delivery into the malignant tissues. The latter approach includes on the one hand two strategies to enhance tumor deliveryargeting (augmented uptake via folate receptor by attachment of folic acid and targeting of bone metastases by bisphosphonate groups) and on the other hand two approaches for evading drug resistance mechanisms (inhibition of efflux pumps by linking to ethacrynic acid and phenoxazines). To gain more insights into the intracellular distribution of Triapine, the synthesis of fluorescent and/or radioactively labeled derivatives is planed. From this extended panel of new thiosemicarbazones lead compounds will be selected for further evaluation of the mechanisms underlying anticancer activity using diverse molecular and cell biological assays. A special focus will be on the impact of clinically relevant drug resistance mechanisms. Finally, the most promising drug candidates will be tested for in vivo anticancer activity in human xenotransplantation models using SCID mice. Taken together, this project will deliver several novel thiosemicarbazone-based drugs rationally modified for enhanced targeting especially of solid tumors. Moreover, the generated data generally should increase of knowledge on the molecular mechanismsargets underlying the anticancer activity of thiosemicarbazone anticancer agents.

Thiosemicarbazones are a promising anticancer substance class targeting important metabolic cancer pathways. Based on their enhanced proliferation, cancer cells are generally hypersensitive to substances targeting DNA synthesis. Accordingly, the lead thiosemicarbazone Triapine is one of the strongest inhibitors of ribonucleotide reductase, an enzyme essential for DNA synthesis. However, despite the promising activity against leukemia, clinical studies in solid tumors with Triapine widely failed. Thus, we aimed in this project to better understand the reasons for this failure and to develop strategies to improve the activity of thiosemicarbazones also against solid tumors. In detail our experiments focused on 1) a better understanding of the mode-of-action of thiosemicarbazones; 2) the generation of novel derivatives of Triapine with enhanced activity and specific targeting to the cancer tissue; 3) to elucidate mechanism of resistance against these drugs and - based on the gained knowledge - to develop novel strategies for resistance circumvention. During the course of the project we have generated novel key insight regarding the anticancer activity of thiosemicarbazones and targeted all aims mentioned above. Most importantly, we found by extended in vitro and in vivo analyses that both a very rapid clearance from the circulation and an intense induction of broad spectrum drug resistance might be major factors contributing to failure of Triapine therapy in the clinic. With regard to resistance development, both massive induction of drug efflux pumps and alterations in unexpected cellular signaling pathways synergistically contributed to resistance failure. These observations are currently used to establish novel treatment strategies based on innovative drug combination schemes involving thiosemicarbazones. Additionally, we have synthesized and extensively characterized multiple novel thiosemicarbazone derivatives including metal complexes and compounds carrying targeting moieties to deliver the drugs specifically to the malignant tissues. While some strategies turned out to be inappropriate (like linkage with biotin or drug resistance-reversing agents), several others turned out to be highly promising. Thus, systematic methylation of the thiosemicarbazone core did not only massively alter anticancer activity but also vulnerability by drug resistance mechanism. Moreover, copper and iron complexes and bone-targeting derivatives of Triapine were characterized as interesting anticancer compounds. Thus, this project offers novel strategies for successful application of this compound class in anticancer therapy.Within the frame of this project, one PhD student and one diploma student successfully performed their theses, two post Doc scientists successfully developed their careers (one university position, one habilitation) and the PI received a full professorship. Moreover, this interdisciplinary project significantly contributed to the establishment of the interuniversity research platform for Translational Cancer Therapy Research as a permanent cooperative structure between the Medical University and the University Vienna.