Proceedings of the 36 th European Peptide Symposium Michal Lebl (Editor) European Peptide Society, 2022 https://doi.org/10.17952/36EPS.2022.151 Development of Pancreatic Tumor Specific Daunomycin – Peptide Conjugates Using Homing Peptides Selected by Phage Display Technique Levente Endre Dókus 1 , Eszter Lajkó 3 , Zsófia A. Szász 3 , Diána Vári-Mező 3 , Angéla Takács 3 , László Kőhidai 3 , and Gábor Mező 1,2 1 ELKH-ELTE Research Group of Peptide Chemistry, Budapest, Hungary; 2 Eötvös Loránd Universit, Faculty of Science, Institute of Chemistry, Budapest, Hungary; 3 Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary Introduction Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most dangerous cancerous diseases leading to high mortality [1]. This tumor type is only 3-4% of all newly diagnosed cancer cases but the mortality of this disease is more than 80% [2]. Furthermore, the average 5-years survival rate is not more than 10% [3], because approximately 80% of the patients are diagnosed with metastatic or inoperable status. Therefore, new approaches for efficient treatment are necessary. Here we report the development of peptide - drug conjugates which contain daunomycin as anticancer drug attached to different homing peptides via oxime bound – a linkage that might be suitable for targeted tumor therapy. In the literature, several peptides can be found that were identified by phage-display technique and recognized PDAC cells with high affinity. However, the efficacy of drug targeting has not been compared. Four of them ((I) SYENFSA [4], (II) IVRGRVF [4], (III) PFWSGAV [5], (IV) TMAPSIK [6]) were selected for this study. Based on our previous results four-members conjugate families were designed. Linear and branched structures were synthesized using various enzyme labile spacers for enhanced drug release. Results and Discussion For targeting of PDAC cells, sixteen different daunomycin - peptide conjugates were designed and synthesized using the above mentioned four homing moieties (Table 1). The synthesis of conjugates was carried out by solid phase peptide synthesis, using Fmoc/ t Bu technique. To the N-terminal α-amino group isopropylidene-protected aminooxyacetyl-group was attached as a linker between the homing part and the payload. The aminooxyacetyl peptide derivatives were cleaved from the resin and the crude products were purified by RP-HPLC. For identification of products ESI-MS was used in all cases. After the cleavage of the isopropylidene-protecting group with 0.2M NH4OAc buffer solution (pH 5.0), daunomycin was coupled to the aminooxyacetyl moiety forming oxime bond (Figure 1). In the case of eight conjugates cathepsin B enzyme labile spacer was built into the sequences between the homing peptide and the aminooxy moiety that may enhance the intracellular (lysosomal) degradation and the release of the active drug-containing metabolites. In cases of apolar homing peptide sequences (1, 2, 3) LRRY segment was built in as spacer but the more polar 4 sequence encouraged the application of GFLG spacer [7]. The in vitro cytotoxic effect of conjugates was investigated on PANC-1 pancreatic cancer cells by an impedimetric technique, xCELLigence System. The effect of them was characterized also on further three cell lines as control (colon cancer (Colo-205); melanoma (A2058) and non-small cell lung cancer (EBC-1)) for characterization their selectivity using Alamar blue test. 151 151 151 151 151