Suchergebnisse

Simple Summary: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Serrated adenocarcinoma (SAC) has been recently recognized by the WHO as a histological CRC with bad prognosis. Consistent with previous evidence, our group identified Fascin1 as a protein directly related to the invasiveness of tumor cells, overexpressed and positively correlated with worse survival in various carcinomas, including SAC. Therefore, Fascin1 has emerged as an ideal target for cancer treatment. In the present study, virtual screening has been carried out from a library of 9591 compounds, thus identifying the FDA-approved anti-retroviral raltegravir (RAL) as a potential Fascin1 blocker. In vitro and in vivo results show that RAL exhibits Fascin1-binding activity and Fascin1-dependent anti-invasive and anti-metastatic properties against CRC cells both in vitro and in vivo. Abstract: Background: Fascin1 is the key actin-bundling protein involved in cancer invasion and metastasis whose expression is associated with bad prognosis in tumor from different origins. Methods: In the present study, virtual screening (VS) was performed for the search of Fascin1 inhibitors and RAL, an FDA-approved inhibitor of human immunodeficiency virus-1 (HIV-1) integrase, was identified as a potential Fascin1 inhibitor. Biophysical techniques including nuclear magnetic resonance (NMR) and differential scanning fluorimetry (DSF) were carried out in order to confirm RAL as a Fascin1 blocker. The effect of RAL on actin-bundling activity Fascin1 was assessed by transmission electron microscopy (TEM), immunofluorescence, migration, and invasion assays on two human colorectal adenocarcinoma cell lines: HCT-116 and DLD-1. In addition, the anti-metastatic potential of RAL was in vivo evaluated by using the zebrafish animal model. Results: NMR and DSF confirmed in silico predictions and TEM demonstrated the RAL-induced disorganization of the actin structure compared to control conditions. The protrusion of lamellipodia in cancer cell line overexpressing Fascin1 (HCT-116) was abolished in the presence of this drug. By following the addition of RAL, migration of HCT-116 and DLD-1 cell lines was significantly inhibited. Finally, using endogenous and exogenous models of Fascin1 expression, the invasive capacity of colorectal tumor cells was notably impaired in the presence of RAL in vivo assays; without undesirable cytotoxic effects. Conclusion: The current data show the in vitro and in vivo efficacy of the antiretroviral drug RAL in inhibiting human colorectal cancer cells invasion and metastasis in a Fascin1-dependent manner. ; Instituto de Salud Carlos III Spanish Government ; European Commission PI15/00626 PI18/00144 ; European Commission's Corbel Program PID-3630 2334 2428 ; Spanish Ministry of Economy and Competitiveness MINECO CTQ2017-87974-R ; Fundación Seneca 20988/PI/18 20646/JLI/18 ; UCAM FPI05-UCAM/17 ; Junta Provincial Murcia Predoctoral Asociación Española contra el Cáncer (AECC) PRDMU19002

This research has been financed by grants BIO2016-78746-C2-1-R and PID2020-112895RB-100 from the Spanish Ministry of Education and Science (I.L.) including AEI/FEDER EU funds. R.N.H. was funded in part by National Institutes of Health grants AI138052 and AI138630. M. I.B. and J.M.C. were recipients of a research contract from the Spanish Ministry of Education and Science. F.C. was funded by a predoctoral fellowship from the Andalusian Government P10-CVI-5915. J.M.C. ac-knowledges a reincorporation research contract from the University of Granada. We thank Dr. Sachdev Sidhu for his invaluable assistance setting up the phage display techniques in our laboratory. We also thank the support of the C.I.C. of the University of Granada. ; The recognition of PPxY viral Late domains by the third WW domain of the human HECT-E3 ubiquitin ligase NEDD4 (NEDD4-WW3) is essential for the budding of many viruses. Blocking these interactions is a promising strategy to develop broad-spectrum antivirals. As all WW domains, NEDD4-WW3 is a challenging therapeutic target due to the low binding affinity of its natural interactions, its high conformational plasticity, and its complex thermodynamic behavior. In this work, we set out to investigate whether high affinity can be achieved for monovalent ligands binding to the isolated NEDD4-WW3 domain. We show that a competitive phage-display set-up allows for the identification of high-affinity peptides showing inhibitory activity of viral budding. A detailed biophysical study combining calorimetry, nuclear magnetic resonance, and molecular dynamic simulations reveals that the improvement in binding affinity does not arise from the establishment of new interactions with the domain, but is associated to conformational restrictions imposed by a novel C-terminal -LFP motif in the ligand, unprecedented in the PPxY interactome. These results, which highlight the complexity of WW domain interactions, provide valuable insight into the key elements for high binding affinity, of interest to guide virtual screening campaigns for the identification of novel therapeutics targeting NEDD4-WW3 interactions. ; Spanish Government BIO2016-78746-C2-1-R PID2020-112895RB-100 AEI/FEDER EU funds AI138052 AI138630 ; United States Department of Health & Human Services ; National Institutes of Health (NIH) - USA P10-CVI-5915 ; German Research Foundation (DFG) ; University of Granada