Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. Among several receptors upregulated in cancer cells, the gastrin-releasing peptide receptor (GRP-R) has recently emerged as a promising target for cancer imaging, diagnosing and treatment due to its overexpression on cancerous tissues such as breast, prostate, pancreatic and small-cell lung cancer. Herein, we report on the in vitro and in vivo selective delivery of the cytotoxic drug daunorubicin to prostate and breast cancer, by targeting GRP-R. Exploiting many bombesin analogues as homing peptides, including a newly developed peptide, we produced eleven daunorubicin-containing peptide–drug conjugates (PDCs), acting as drug delivery systems to safely reach the tumour environment. Two of our bioconjugates revealed remarkable anti-proliferative activity, an efficient uptake by all three tested human breast and prostate cancer cell lines, high stability in plasma and a prompt release of the drug-containing metabolite by lysosomal enzymes. Moreover, they revealed a safe profile and a consistent reduction of the tumour volume in vivo. In conclusion, we highlight the importance of GRP-R binding PDCs in targeted cancer therapy, with the possibility of further tailoring and optimisation.

Assembly and disassembly of protein–protein complexes needs to be dynamically controlled and phosphoswitches based on linear motifs are crucial in this process. Extracellular signal–regulated kinase 2 (ERK2) recognizes a linear‐binding motif at the C‐terminal tail (CTT) of ribosomal S6 kinase 1 (RSK1), leading to phosphorylation and subsequent activation of RSK1. The CTT also contains a classical PDZ domain‐binding motif which binds RSK substrates (e.g. MAGI‐1). We show that autophosphorylation of the disordered CTT promotes the formation of an intramolecular charge clamp, which efficiently masks critical residues and indirectly hinders ERK binding. Thus, RSK1 CTT operates as an autoregulated phosphoswitch: its phosphorylation at specific sites affects its protein‐binding capacity and its conformational dynamics. These biochemical feedbacks, which form the structural basis for the rapid dissociation of ERK2‐RSK1 and RSK1‐PDZ substrate complexes under sustained epidermal growth factor (EGF) stimulation, were structurally characterized and validated in living cells. Overall, conformational changes induced by phosphorylation in disordered regions of protein kinases, coupled to allosteric events occurring in the kinase domain cores, may provide mechanisms that contribute to the emergence of complex signaling activities. In addition, we show that phosphoswitches based on linear motifs can be functionally classified as ON and OFF protein–protein interaction switches or dimmers, depending on the specific positioning of phosphorylation target sites in relation to functional linear‐binding motifs. Moreover, interaction of phosphorylated residues with positively charged residues in disordered regions is likely to be a common mechanism of phosphoregulation.Database: Structural data are available in the PDB database under the accession numbers 5N7D, 5N7F and 5N7G. NMR spectral assignation data are available in the BMRB database under the accession numbers 27213 and 27214.

Among various homing devices, peptides containing the NGR tripeptide sequence represent a promising approach to selectively recognize CD13 receptor isoforms on the surface of tumor cells. They have been successfully used for the delivery of various chemotherapeutic drugs to tumor vessels. Here, we report on the murine plasma stability, in vitro andin vivoantitumor activity of our recently described bioconjugates containing daunorubicin as payload. Furthermore, CD13 expression of KS Kaposi’s Sarcoma cell line and HT-29 human colon carcinoma cell line was investigated. Flow cytometry studies confirm the fast cellular uptake resulting in the rapid delivery of the active metabolite Dau = Aoa-Gly-OH to tumor cells. The increased in vitro antitumor effect might be explained by the faster rearrangement from NGR toisoDGR in case of conjugate2(Dau = Aoa-GFLGK(c[NleNGRE]-GG)-NH₂) in comparison with conjugate1(Dau = Aoa-GFLGK(c[KNGRE]-GG)-NH₂). Nevertheless, results indicated that both conjugates showed significant effect on inhibition of proliferation in the primary tumor and also on blood vessel formation making them a potential candidate for targeting angiogenesis processes in tumors where CD13 and integrins are involved.