Construction Of Cell-penetrating Peptide?Arg?₉ And SH2 Superbinder Fusion Proteins And The Study Of Its Anti-tumor Related Signal Pathways

Melanoma,which can be cured by surgery in the early stage but the prognosis is poor,accounts for about 80%of skin cancer deaths.This is largely because metastatic malignancy is refractory to conventional therapies.Gene mutations,mostly BRAF mutations,are present in over half of all human melanomas.Although patients with BRAF-mutant are highly sensitive to small molecule inhibitors,like Vemurafenib and Dabrafenib,most patients display partial responses to clinical treatment,which can eventually worsen conditions or cause death due to drug resistance within 6 to 12months.Moreover,patients that lack gene mutations have few effective therapeutic methods.These patients are associated with unique risk factors,pathophysiologic,clinical,and prognostic features that differ from those related to BRAF tumors.Thus,it is necessary and urgent to address the problems of drug resistance to inhibitors with BRAF-mutant and lacking appropriate therapies for patients without specific mutations.Src Homology 2?SH2?domains specifically recognize phosphorylated tyrosine and mediate cell signal transduction.The“superbinder”SH2 domain,with triple-point mutants Thr8Val/Cys10Ala/Lys15Leu,abbreviated SH2 superbinder,which could bind pY-containing peptides with much stronger affinity than natural SH2 domains or traditional anti-pY antibody?4G10?.Conventional drugs are generally directed against a single gene or protein and most of melanoma patients develop drug resistance eventually.SH2 superbinder,strongly binding with diverse pY sites to block related signal transduction pathways,can recognize other sites instead,even if mutation alters the pY site.Therefore,SH2 superbinder can achieve the purpose of killing cancer cells.These advantages might resolve problems of drug resistance due to gene mutation and lack of suitable targeting drugs described above.However,it is urgent to settle the problem of transporting SH2 superbinder across cell membrane barriers.Cell penetrating peptides?CPPs?,having ability to translocate across the plasma membranes,are confined to short sequences of less than 20 amino acids.CPPs have several advantages over conventional techniques on cellular delivery because they are efficient for a variety of cells,and have a potential therapeutic application.Nona-arginine?Arg?₉,as one kind of CPPs,has been applied efficaciously to translocate across the cell membrane and deliver large cargo molecules such as peptides,proteins,and oligonucleotides into cells with no severe side effects,which has a prospect of wide application and a development potential in drug delivery.Aptamers are single-stranded oligonucleic acid molecules that are screened via SELEX in random oligonucleotide libraries.They have high affinity with the target molecule.Compared to conventional antibodies that specifically recognize cell surface markers,aptamers have the advantages of strong specificity,wide range of receptors,low cost,and lack of immunogenicity.Studies have shown that nucleolin are transferred to the surface of cell membrane in a variety of cancer cells,and the aptamer AS1411screened for nucleolin can target these cells.As the first aptamer to enter clinical tumor tests,AS1411 showed a significant effect of inhibiting tumor cell proliferation in vitro.In order to make full use of the advantages of SH2 superbinder,cell-penetrating peptides,and aptamers,we gradually generate the“Cell-penetrating peptide-SH2superbinder-Aptamer”conjugate as a novel target drug candidate.The conjugate can specifically recognize and bind cancer cells through the aptamer,and convey SH2superbinder into cells utilizing the cell-penetrating peptides,so that the SH2superbinder could combine with many pY-containing proteins in cells,thereby affecting multiple signaling pathways related to proliferation,migration and apoptosis.This innovative method may ensure multi-targets as well as specificity,effectively delaying the development of drug resistance and side effects on normal cells.