Agonist and antagonist peptide ligands of CXCR4 receptor as marker for the anticancer drugs or diagnostic agents delivery
Creators: name: Monfregola, Luca
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2010

description: Chemokines are a family of small low molecular weight secreted cytokines that regulate cell migration by activating a set of G-protein-coupled receptors (GPCRs). Beside the physiologic role, chemokines and their receptors participate in numerous disease states, including HIV/AIDS, asthma, autoimmune diseases, and cancer. Over expression of CXCR4 receptor and over production of its only ligand, the chemokine CXCL12 (also called stromal cell-derived factor-1, SDF-1), was described in brain neoplasm, neuroblastoma cells, colorectal cancer, prostate cancer, melanoma,renal cell cancer, ovarian cancer, and others. Because it directs stem-cell homing and participates in nearly every aspect of cancer progressions, growth, metastasis and neovascularization, the CXCL12/CXCR4 signaling axis is of increasing interest for drug discovery. Among the CXCR4 inhibitors there are two major classes of CXCR4 antagonists, small-molecule antagonists (AMD3100 and its analogs) and peptidomimetics (T140 and its analogs).  
As for other chemokines, structure-activity studies of SDF-1 have shown the critical role of the N-terminal region for both receptor binding and activation and in particular the residues 1-8 and 12-17, these last being located in the loop region.  
vMIP-II, a CC chemokine-like protein encoded by Kaposi's sarcoma-associated herpesvirus, binds and blocks chemokine receptors belonging to the CXC, CC and XC class, such as CCR1, CCR2. CCR5, CCR8, XCR1 and CXCR4. As for SDF-1, the N-terminus and the N-loop are essential for receptor binding. In particular it was demonstrated that the N-terminus alone, encompassing residues 1-10, is sufficient for binding and antagonizing CXCR4 receptor. Therefore in this study we have focused on a sequence-structure comparison between the N-terminal regions of SDF-1 and vMIP-II, with the aim of looking for a possible common motif responsible for the binding to CXCR4. On the basis of this comparison, several cyclic peptides containing a putative common motif have been designed, studied by molecular dynamics simulations and then synthesized. The peptides differ in: a) nature of the aromatic residues; b) sequence sense; c) N- and C-termination, as all combinations of free, single- and double-protected (by acetylation and amidation) termini were tested on selected peptides; d) possible elongation at either peptide termini by a Arg-Ala sequence. Their activity has been tested by different essays addressing some of the many physiological and pathological functions of CXCR4 receptor:  Binding through flow cytometry, modulation of  intracellular Ca2+ release, modulation of cell migration in presence or without specific ligand SDF-1 and Modulation of P-Erk activation. 
The pattern of biological responses elicited by these peptides was, heterogeneous demonstrating agonism, antagonism and no interference for the same peptide in different assays. These results cannot be explained simple models interaction receptor activation/inhibition and suggest more complex scenario considering  direct CXCL12-peptide interactions and/or influence on symmetry, stoichiometry or structural variations of the homo- or hetero-oligomeric state of CXCR4 receptor. Among the peptides with consistent inhibitory activity on CXCR4 four peptides were identified: peptides C1, C16, C17 and C18. In particular peptide C16 is inhibitor in all the evaluated in vitro assays performed. Based on these results, the in vivo effect on metastases formation was tested. The peptides (C1, C16 and C18) significantly inhibited melanoma metastases and preliminary results showed that renal cancer cells xenograft  SN12C-pEGFP was significantly reduced in growth in the presence of peptides C1, C16 and C18.  
As a subsequent step, it was evaluated the possibility to develop peptide C18 derivative bearing a chelating agent able to coordinate radioactive metals for applications in cancer diagnosis by nuclear medicine techniques. In order to investigate the chelating agent site of linkage which does not interfere on the peptide-receptor interaction, two diethylentriaminopentacetic acid (DTPA)-C18 conjugates were synthesized:  one carrying DTPA moiety covalently bound to petide N-terminus (C20) and one carrying the chelating moiety DTPA covalently bound to the epsilon-NH2 of a lysine residue which replaced  Ala2 of C18 (C19).  
Before performing nuclear medicine imaging studies, C19 and C20 were evaluated for their ability to inhibit the specific antibody binding  to CXCR4. Both conjugates exhibited a reduced inhibiting activity, compared with that exerted by the only peptide sequence (C18). These results highlighted that the introduction of the chelating agent on the N-terminus, as well as on the lysine epsilon-NH2 group, can affect the binding process with the receptor target. We are currently exploring other peptide positions where to anchor the DTPA moiety. 
Since the pharmacophoric motif of the most active peptide sequence (C18) is characterized by aromatic rings and idrofobic residues, it is possible to project peptidomimetics in order to modulate the aromatic rings distance from the peptide backbone. In particular, Phe3 and Phe4 residues of C18 can be replaced by aminobenzilic derivatives, such as Nε-benzylated Lys and its shorter homologues, in order to evaluate the influence of  the more flexible pharmacoforic motif on the binding process. The final goal has been to  introduce conformational constraint into the peptide sequence by which it would be possible to stabilize the so-called 'bioactive' conformation, that is the peptide conformation required for receptor binding and activation. 
On these basis, we focused on an alternative and more practical synthetic strategy in solution and in solid phase in order to obtain modified amino acids  to be used as a building block in peptidomimetic synthesis. In particular  we performed the alkylation reaction on  several  Fmoc-amino acids, protected on their side chain, (Fmoc-Lys(P)-OH, Fmoc-Orn(P)-OH, Fmoc-Dab(P)-OH Fmoc-Dap(P)-OH) only in presence of 4 Ǻ molecular sieves and alkyl halides. This methodology was validated for different amino protecting groups, but the best results were obtained by using o-Ns protected Fmoc-amino acids. Furthermore, for the majority of the employed halides, the procedure is a one-pot synthesis which avoids the purification after each reaction step. As final step we verified the applicability of building block synthesized by introducing one of them into a peptide sequence using the standard Fmoc-based solid phase protocol.  
Data complied and the methodology developed open new perspectives in obtaining more selective compounds toward different biological pathways involving CXCR4 receptor. In this regard, the forthcoming activity will be focused on the development of peptidomimetics that allow stabilizing conformation required for receptor binding and activation. Moreover, dimers of the most active peptides will be also synthesized and tested for their biological activity, in order to evaluate any potential improvement of it compared with the activity exerted by the corresponding monomeric peptide sequence.