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Angiogenesis, the process whereby new capillaries are formed by outgrowth from existing microvessels, is required for tumor growth and metastasis. The transmembrane cell-surface receptor ?V?3 has recently received increasingly attention, because of the critical role in tumor associated angiogenesis and metastasis formation . The restricted expression of integrin ?V?3 during tumor growth, invasion, and metastasis presents an interesting target for both detection and treatment of solid tumors. Targeting ?V?3 with radiolabelled ligands may provide information about the receptor status and enable the planning and the monitoring of therapeutic approaches. Recently we developed a novel ?V?3 antagonist that showed a high selectivity for the receptor. Adhesion assays, competitive binding assays and cross-linking experiments performed in human erythroleukemia K562 cells, stably cotransfected with cDNA of ?v or ?IIb and ?3, demonstrated the high selectivity for ?V?3 integrin. Starting from these evidences, RGDechi was covalently bound to the chelating agent DTPA able to give stable complexes of radionuclides, such as 111In and 90Y . The final goal has been to obtain a radiolabelled compound to be used in nuclear medicine as a diagnostic and therapeutic agent. In particular DTPA-RGDechi has been labeled with 111In and used in SPECT for diagnostic purpose. Moreover, the ligand RGDechi has been labeled with 18F for micro-PET in order to evaluate the use of this peptide for in vivo imaging.

Aim. The alpha(v)beta(3) integrin is a cell adhesion receptor involved in angiogenesis, tumor cell migration and metastatic dissemination. The tripeptide sequence RGD binds to alpha(v)beta(3) but also interacts with other integrins. We developed a novel cyclized RGD pentapeptide covalently linked by a spacer to an echistatin domain that showed a high selectivity for alpha(v)beta(3) integrin ( J Med Chem 2006, 49:3416-3420). Materials and Methods. In the present study we evaluated this chimeric RGD peptide (RGDechi) in human erythroleukemia K562 cells, stably cotransfected with cDNA of alpha(v) or alpha(IIb) and beta(3) or beta(5) subunits by adhesion assays, competitive binding assays and cross-linking experiments. RGDechi was then conjugated with DTPA and labeled with 111In for SPECT imaging whereas a one-step procedure was used for labeling the chimeric peptide with 18F for PET imaging. K562 cells overexpressing alpha(v)beta(3) or alpha(v)beta(5) were subcutaneously injected into opposite flanks of individual nude mice and allowed to grow up to 0.5 cm in size. Alternatively, U87MG human glioblastoma cells and A431 human epidermoid cells endogeneously expressing high levels of alpha(v)beta(3) and alpha(v)beta(5), respectively, were used to develop xenografts in nude mice. Imaging studies were then performed using both SPECT and microPET. Results. Adhesion assays showed that the chimeric RGDechi was able to inhibit adhesion of alpha(v)beta(3) overexpressing cells but not alpha(IIb)beta(3) and alpha(v)beta(5) overexpressing clones to native ligands. Nude mice bearing K562 and U87MG tumor xenografts, both overexpressing alpha(v)beta(3), showed a high tumor uptake of 111In-labeled and 18F-labeled RGDechi assessed by SPECT and microPET, respectively. No tumor uptake of radiolabeled RGDechi could be observed in K562 and A431 tumor xenografts overexpressing alpha(v)beta(5). Conclusion. Our findings indicate that chimeric RGDechi is suitable for in vivo selective alpha(v)beta(3) receptor imaging.

Background: In malignant melanoma (MM), overexpression of alfavbeta3 integrin is linked to a more metastatic phenotype. Development of anti-alfavbeta3 agents able to counteract melanoma progression would be helpful for disease treatment. A new selective ligand of alfavbeta3, RGDechi-hCit, has anti-angiogenic properties against endothelial cells in angiogenesis animal models. Aim of this study was to evaluate the in vitro effects of the RGDechi-hCit peptide on MM cell lines. Material and Methods: Cytofluorimetric analysis characterized cell surface expression of alfavbeta3 integrin on seven MM cell lines. Cell proliferation, adhesion, and migration assays were carried out using the alfavbeta3-antagonist RGDechi-hCit. Results: Proliferation was not significantly inhibited by RGDechi-hCit, though striking morphological changes were detected in MM cell lines highly expressing alfavbeta3. Conversely, assays on fibronectin-coated plates showed a significant RGDechi-hCit dose-dependent inhibitory effect on both adhesion and migration. Conclusions: Data demonstrate anti-adhesion and anti-migration, but not anti-proliferative, activities of RGDechi-hCit against MM cells.

York (Regno Unito)

Pox virus Zinc/Bric-à-brac, Tramtrack and Broad (POZ/BTB) is a widespread domain detected in proteins involved in a variety of biological processes. Human genome analyses have unveiled the presence of POZ/BTB domain in a class of proteins (KCTD) whose role as important players in crucial biological processes is emerging. The development of new molecular entities able to interact with these proteins and to modulate their activity is a field of relevant interest. By using molecular modeling and literature mutagenesis analyses, we here designed and characterized a peptide that is able to interact with submicromolar affinities with two different members (KCTD11 and KCTD5) of this family. This finding suggests that the tetrameric KCTD11 and the pentameric KCTD5 are endowed with a similar cavity at the subunit-subunit interface deputed to the Cul3 binding, despite their different oligomeric states.

Purpose: To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg- Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpavbeta3 from alpavbeta5 integrin, thus allowing the in vivo selective visualization of alpavbeta3 expression by single-photon and positron emission tomography (PET) imaging. Experimental Design: The chimeric peptide was preliminarily tested for inhibition of alphavbeta3-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpavbeta3 (Kalpavbeta3 ) or alphavbeta5 (Kalpavbeta5) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and la- beled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-termi- nal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpavbeta3 , Kalpavbeta5, U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Results: Adhesion and competitive binding assays showed that the novel chimeric pep- tide selectively binds to alpavbeta3 integrin and does not cross-react with alpavbeta5. In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiola- beled chimeric peptide selectively localizes in tumor xenografts expressing alpavbeta3 and fails to accumulate in those expressing alpavbeta5 integrin. When 18F-labeled truncated derivative was used for PET imaging, alpavbeta3 - and alpavbeta5-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Conclusion: Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alpavbeta5 integrin, allows highly selective alpavbeta3 expression imaging and monitoring. (Clin Cancer Res 2009;15(16):5224-33)

We report here the synthesis and molecular structure in the solid state of fully protected tripeptides containing Cα,α-diphenylglycine (Dph), namely Z-Aib-Dph-Gly-OMe (Aib: Cα,α-dimethylgrycine) and Bz-Dph-Dph-Gly-OMe. The molecular conformation around the Dph residue, containing two bulky substituents, is fully extended, while the Aib residue, containing two smaller groups on the Cα atom, adopts the typical 310/α-helical conformation. Gly residues, without substituents on the Cα atom, show different conformational preferences. Each residue seems to behave, from a conformational point of view, independently from the presence of the other residues, and thus mixed local conformations (folded and extended) are present in the crystals. The nonconventional peptide synthesis, using the Ugi reaction, is also reported.