DESIGN, SYNTHESIS AND CYTOTOXICITY STUDY OF NOCARDIOTIDE ANALOGS AS RADIOPHARMACEUTICAL LIGAND CANDIDATE BASED ON PEPTIDES FOR CANCER DETECTION AND THERAPY

• Main Author: Juwita Sugiharti, Rizky
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/76388
• Institution: Institut Teknologi Bandung

Accurate cancer detection at an early stage is imperative as a prerequisite of therapy to stop its spread and increase patients’ survival. Somatostatin 2 receptor (SSTR2), an over-expressed peptide receptor in cancer tissues is a potential molecular target of cancer imaging to detect cancer. Radiopeptide targeting the SSTR2 showed promising potential in cancer detection at an early stage. Nocardiotide, a cyclic hexapeptide consists of the amino acids cyclo-Trp1-Ile-Trp2-LeuVal-Ala isolated from Nocardiopsis sp. has significant cytotoxicity against cancer cells which makes this peptide suitable for further development as the main compound of radiopeptide. This study aims to design a stable and effective nocardiotide-based radiopeptide without omitting its affinity to SSTR2 for cancer detection and therapy. In-silico study was conducted to design nocardiotide analogs through structure modification and molecular interactions assessment with SSTR2. Five nocardiotide analogs that are going to be labeled with technetium-99m an indirect labeling method were designed by replacing its valine amino acids with NH2-containing amino acids, namely lysine, arginine, histidine, asparagine, and glutamine (cWIWLKA/cWIWLRA/cWIWLHA/cWIWLNA/cWIWLQA). Next, the interaction assessment was done based on the technetium metal structure model of [ 99mTc/EDDA/HYNICpeptide] and [ 99mTc/tricine/HYNIC-peptide]. In addition, six nocardiotide analogs that are going to be labeled with iodine-131 by a direct labeling method were designed by inserting histidine and tyrosin as radiolabeling targets, replacing valine, leucine, and isoleucine (cWHWLVA/ cWIWHVA/ cWIWLHA/ cWIWLYA/ cWIWYVA dan cWYWLVA). The interaction assessment was done based on the mono-iodinated [ 131I-peptide] and di-iodinated peptide peptide [131I2- peptide] structure model. Based on the in-silico study results, the nocardiotide analogs were synthesized and then used in an in-vitro study against HeLa cancer cells to determine its IC50. Preliminary labeling of nocardiotide analogs with a radioisotope was then conducted following the result from in-silico and in-vitro studies. Based on the results of in silico studies, the nocardiotide-based radiopeptide labeled by 99mTc or 131I showed a good affinity value with hydrogen bonding in a SSTR2 residues of Q102, Q126, D122, C193, T212 and N276. Nocardiotide analogs of cWIWLKA, cWIWLNA, and cWIWLYA were selected for further synthesis and development. Linear hexapeptides of WIWLKA, WIWLNA, and WIWLYA were synthesized by solid phase peptide synthesis method at 2-chlorotrityl chloride resin with Fmoc strategy. Characterization by TOF-ESI-MS at the protected crude linear peptide yielding an expected linear peptide suitable with the preferred ionic molecule, with the value of m/z [M+H]+ 1116,6583 (rendement, 73,7%), m/z [M+H]+ 1244,5322 (rendement, 34,13%), and [M+H]+ 1107,6298 (rendement, 52.90%) for WIWLKA, WIWLNA, and WIWLYA, respectively. Linear hexapeptide cyclization using PyBOP reagent using DMF and DIPEA base solvent was confirmed by the presence of an ionic molecule peak m/z [M+H]+ 798,4608 (rendement, 22.40%), [M+K]+ 822,9913 (rendement, 15.88%), and [M+Na]+ 855,4332 (rendement, 25.60%) for cWIWLKA, cWIWLNA, and cWIWLYA, respectively. Structural analysis using 13C-NMR and 1H-NMR showed that the structures of the three analogues were confirmed according to the desired compounds. In-vitro test of synthesized nocardiotide analogs against HeLa cancer cells resulting IC50 value of 121.2 ?g/mL, 128,2 ?g/mL, and 165,6 ?g/mL for cWIWLKA, cWIWLNA, and cWIWLYA, respectively. The results of this study indicate the inhibitory activity and sensitivity of the three analog compounds against cancer cells. The cWIWLKA was selected as a 99mTc-based radiopeptide designed with a bifunctional chelator HYNIC-Boc. Peptide conjugation reaction at HYNIC showed an ionic molecule peak of HYNICcWIWLKA pada m/z [M+H]+ 1033,6199 that is suitable to the target ionic molecule target. Next, the cWIWLYA was selected as an iodine-131-based radiopeptide that was labeled by the cold labeling method. It showed di-iodinated radiopeptide at m/z [M+Na]+ 1107,1138 and monoiodinated radiopeptide at m/z 958 [M]. As a conclusion, nocardiotide-based radiopeptide has the potential to further be developed as a radiopharmaceutical for cancer diagnosis and treatment.