Determination of the binding epitope of RGD-peptidomimetics to \u3b1v\u3b23 and \u3b1IIb\u3b23 integrin-rich intact cells by NMR and computational studies

NMR experiments (transferred NOE and Saturation Transfer Difference) were used to shed light on the binding epitope of RGD peptidomimetics 1-3 with integrins \u3b1v\u3b23 and \u3b1IIb\u3b23, expressed on the membrane of ECV304 bladder cancer cells and human platelets, respectively. The NMR results were supported by docking calculations in the active sites of \u3b1v\u3b23 and \u3b1IIb\u3b23 integrin receptors and were compared to the results of competitive \u3b1v\u3b23 receptor binding assays and competitive ECV304 cell adhesion experiments. While cis RGD ligand 1 interacts mainly with the \u3b1 integrin subunit through its basic guanidine group, trans RGD ligands 2 and 3 are able to interact with both the \u3b1 and \u3b2 integrin subunits via an electrostatic clam

Synthesis and Biological Evaluation (in Vitro and in Vivo) of Cyclic RGD Peptidomimetic - Paclitaxel Conjugates Targeting Integrin alphaVbeta3

A small library of integrin ligand - Paclitaxel conjugates 10-13 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the Paclitaxel-RGD constructs 10-13 inhibited biotinylated vitronectin binding to the purified alphaVbeta3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of Paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin alphaVbeta3, making them attractive to be tested in in vivo models. Cyclo[DKP-f3-RGD]-PTX 11 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 11 exhibited a superior activity than Paclitaxel, despite the lower (ca. half) molar dosage used

Rational Design of Antiangiogenic Helical Oligopeptides Targeting the Vascular Endothelial Growth Factor Receptors

Tumor angiogenesis, essential for cancer development, is regulated mainly by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which are overexpressed in cancer cells. Therefore, the VEGF/VEGFR interaction represents a promising pharmaceutical target to fight cancer progression. The VEGF surface interacting with VEGFRs comprises a short α-helix. In this work, helical oligopeptides mimicking the VEGF-C helix were rationally designed based on structural analyses and computational studies. The helical conformation was stabilized by optimizing intramolecular interactions and by introducing helix-inducing Cα,α-disubstituted amino acids. The conformational features of the synthetic peptides were characterized by circular dichroism and nuclear magnetic resonance, and their receptor binding properties and antiangiogenic activity were determined. The best hits exhibited antiangiogenic activity in vitro at nanomolar concentrations and were resistant to proteolytic degradation

Synthesis and Biological Evaluation of RGD and isoDGR-Monomethyl Auristatin Conjugates Targeting Integrin (V3)

This work reports the synthesis of a series of small molecule-drug conjugates containing the \u3b1V\u3b23-integrin ligand cyclo[DKP-RGD] or cyclo[DKP-isoDGR], a lysosomally cleavable Val-Ala (VA) linker or an "uncleavable" version devoid of this sequence, and monomethyl Auristatin E (MMAE) or F (MMAF) as cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide-alkyne cycloaddition as key-step. The conjugates were tested for their binding affinity to the isolated \u3b1v\u3b23 receptor, and shown to retain nanomolar IC50 values, in the same range of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with \u3b1v\u3b23 integrin over-expressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess a markedly lower cytotoxic activity compared to the free drugs, which is consistent with an inefficient integrin-mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, cyclo[DKP-isoDGR]-VA-MMAE conjugate has low nanomolar IC50 values in cell viability assays with both cancer cell lines tested (U87: 11.50 \ub1 0.13 nM; M21: 6.94 \ub1 0.09 nM) and is therefore a promising candidate for in vivo experiments.This work reports the synthesis of a series of small-molecule\u2013drug conjugates containing the \u3b1 V \u3b2 3 -integrin ligand cyclo[DKP-RGD] or cyclo[DKP-isoDGR], a lysosomally cleavable Val-Ala (VA) linker or an \u201cuncleavable\u201d version devoid of this sequence, and monomethyl auristatin E (MMAE) or F (MMAF) as the cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide\u2013alkyne cycloaddition as the key step. The conjugates were tested for their binding affinity for the isolated \u3b1 v \u3b2 3 receptor and were shown to retain nanomolar IC 50 values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with \u3b1 v \u3b2 3 integrin overexpressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess markedly lower cytotoxic activity than the free drugs, which is consistent with inefficient integrin-mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, the cyclo[DKP-isoDGR]-VA-MMAE conjugate has low nanomolar IC 50 values in cell viability assays with both cancer cell lines tested (U87: 11.50\ub10.13 nm; M21: 6.94\ub10.09 nm) and is therefore a promising candidate for in vivo experiments

Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery

Borbély AN, Figueras Agustí E, Martins A, et al. Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery. ChemistryOpen. 2019;8(6):737-742.RGD-cryptophycin and isoDGR-cryptophycin conjugates were synthetized by combining peptidomimetic integrin ligands and cryptophycin, a highly potent tubulin-binding antimitotic agent across lysosomally cleavable Val-Ala or uncleavable linkers. The conjugates were able to effectively inhibit binding of biotinylated vitronectin to integrin alphavbeta3, showing a binding affinity in the same range as that of the free ligands. The antiproliferative activity of the novel conjugates was evaluated on human melanoma cells M21 and M21-L with different expression levels of integrin alphavbeta3, showing nanomolar potency of all four compounds against both cell lines. Conjugates containing uncleavable linker show reduced activity compared to the corresponding cleavable conjugates, indicating efficient intracellular drug release in the case of cryptophycin-based SMDCs. However, no significant correlation between the in vitro biological activity of the conjugates and the integrin alphavbeta3 expression level was observed, which is presumably due to a non-integrin-mediated uptake. This reveals the complexity of effective and selective alphavbeta3 integrin-mediated drug delivery

Ex vivo mass spectrometry-based biodistribution analysis of an antibody-Resiquimod conjugate bearing a protease-cleavable and acid-labile linker

Immune-stimulating antibody conjugates (ISACs) equipped with imidazoquinoline (IMD) payloads can stimulate endogenous immune cells to kill cancer cells, ultimately inducing long-lasting anticancer effects. A novel ISAC was designed, featuring the IMD Resiquimod (R848), a tumor-targeting antibody specific for Carbonic Anhydrase IX (CAIX) and the protease-cleavable Val-Cit-PABC linker. In vitro stability analysis showed not only R848 release in the presence of the protease Cathepsin B but also under acidic conditions. The ex vivo mass spectrometry-based biodistribution data confirmed the low stability of the linker-drug connection while highlighting the selective accumulation of the IgG in tumors and its long circulatory half-life