Design of Peptides to Interfere with the RANK-TRAF6 Pathway: an Integrated Approach

• Autor(a) principal: Pereira, Mafalda Inês Apolinário
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/57609
• topic: BSA; Optimized HADDOCK score; Stability; Solid Phase Peptide Synthesis; Purification; Circular Dichorism; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

The RANK-TRAF6 pathway is involved in osteoporosis and in the development of breast cancer. One way to inhibit this pathway is to block TRAF6’s binding to RANK using decoy peptides. It is known by experimental data that a cell-penetrating decoy peptide (cpp) based on TRAF6-binding site mTRANCE-R(1) effectively inhibits this pathway because of its high affinity to TRAF6, whereas the TRAF6-binding site mTRANCE-R(2) does not. One of the goals of this work is to validate by in silico studies the stability of TRAF6 with five peptides, containing TRAF6-binding sites in human (hPEP1, hPEP2, PepH2-hTRANCE1) and mice (mPEP1, mPEP2). The stability of TRAF6 with the different peptides was accessed with two methods. The first method uses the complexes buried surface area (BSA) values, as a higher BSA indicates a higher complex stability. The results obtained with this method are in agreement with the experimental data, suggesting that the peptide with the sequence mTRANCE-R(1) has a higher affinity to TRAF6 than the peptide with the sequence mTRANCE-R(2), indicating that it is more stable with TRAF6. BSA(mTRAF6-mPEP1) > BSA(mTRAF6-mPEP2) and BSA(hTRAF6-hPEP1) > BSA(hTRAF6-hPEP2). BSA(hTRAF6- PepH2-hTRANCE1) > BSA(hTRAF6-hPEP1). The other method uses an optimized HADDOCK score as a tool to predict binding affinity. These results are not in agreement with the literature and the ones determined by BSA studies, failing in validating the stability of TRAF6 with a peptide containing mTRANCE-R(1) and a peptide containing mTRANCE-R(2). Peptides hTRANCE-R(1) and PepH2-hTRANCE1 (>99%) were prepared by manual Fmoc solid phase peptide synthesis followed by HPLC purification. In the PepH2- hTRANCE1 synthesis, besides the desired peptide, we also isolated a side product that was identified as the same peptide sequence with a Valine deletion. This peptide will also be used for future cancer cell studies. The secondary structure of the peptides was analysed by circular dichroism, showing the same fractions of -sheet (0,4) and random coil (0,6) for all.