Synthesis of novel conjugates based on a functionalized cyclo[DKP-isoDGR] integrin ligand and potent cytotoxic agents

Targeted drug delivery is a growing-interest field in cancer therapy as a strategy for overcoming the systemic cytotoxicity associated to traditional chemotherapy. One important approach in this research area is represented by the small molecule-drug conjugates (SMDCs), where the drug-targeting is performed by a low molecular weight ligand (peptide, vitamin or peptidomimetic) connected to a potent warhead through a stable linker. This PhD thesis describes the synthesis and biological evaluation of novel SMDCs containing the functionalized cyclo[DKP-isoDGR] integrin ligand developed by our research group and potent cytotoxic drugs (\u3b1-amanitin, MMAE and MMAF) combined via different linkers and spacers. The conjugates were evaluated for their binding affinity to the isolated \u3b1v\u3b23 receptor and for their antiproliferative activity on cancer cell lines with different levels of \u3b1V\u3b23 expression in order to study the efficacy of the cyclo[DKP-isoDGR] integrin ligand as a vector for tumor drug-delivery

Synthesis of novel conjugates based on a functionalized cyclo[DKP-isoDGR] integrin ligand and potent cytotoxic agents

Targeted drug delivery is a growing-interest field in cancer therapy as a strategy for overcoming the systemic cytotoxicity associated to traditional chemotherapy. One important approach in this research area is represented by the small molecule-drug conjugates (SMDCs), where the drug-targeting is performed by a low molecular weight ligand (peptide, vitamin or peptidomimetic) connected to a potent warhead through a stable linker. This PhD thesis describes the synthesis and biological evaluation of novel SMDCs containing the functionalized cyclo[DKP-isoDGR] integrin ligand developed by our research group and potent cytotoxic drugs (α-amanitin, MMAE and MMAF) combined via different linkers and spacers. The conjugates were evaluated for their binding affinity to the isolated αvβ3 receptor and for their antiproliferative activity on cancer cell lines with different levels of αVβ3 expression in order to study the efficacy of the cyclo[DKP-isoDGR] integrin ligand as a vector for tumor drug-delivery

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

Synthesis of novel conjugates based on a functionalized cyclo[DKP-isoDGR] integrin ligand and potent cytotoxic agents

Targeted drug delivery is a growing-interest field in cancer therapy as a strategy for overcoming the systemic cytotoxicity associated to traditional chemotherapy. One important approach in this research area is represented by the small molecule-drug conjugates (SMDCs), where the drug-targeting is performed by a low molecular weight ligand (peptide, vitamin or peptidomimetic) connected to a potent warhead through a stable linker. This PhD thesis describes the synthesis and biological evaluation of novel SMDCs containing the functionalized cyclo[DKP-isoDGR] integrin ligand developed by our research group and potent cytotoxic drugs (α-amanitin, MMAE and MMAF) combined via different linkers and spacers. The conjugates were evaluated for their binding affinity to the isolated αvβ3 receptor and for their antiproliferative activity on cancer cell lines with different levels of αVβ3 expression in order to study the efficacy of the cyclo[DKP-isoDGR] integrin ligand as a vector for tumor drug-delivery

Synthesis and Biological Evaluation of an isoDGR-Paclitaxel Conjugate Containing a Cell-Penetrating Peptide to Promote Cellular Uptake

Two new Drug Delivery Systems (DDS) cyclo[DKP-isoDGR]-PEG-4-Val-Ala-PTX (2) and cyclo[DKP-isoDGR]-PEG-4-sC18-Val-Ala-PTX (3), containing the cyclo[DKP-isoDGR] integrin ligand and the cytotoxic agent Paclitaxel (PTX), were synthesized to investigate the influence of a PEG-4 chain and of the sC18 cell-penetrating peptide (CPP) on the cellular uptake and the cytotoxicity of the constructs. A double click-reaction strategy was planned, to realize the connection of cyclo[DKP-isoDGR] and PTX to the CPP moiety. Anti-proliferative bioassays were performed on the alpha(V)beta(3)-positive U87 human glioblastoma cell line using a short contact time (15 min) followed by draining, washing of the cells, and re-incubation for 72 h. Compound 3 was significantly more potent (IC50=27.6 mu M) than compound 2 (IC50>100 mu M), and showed a reduced potency loss with respect to PTX (IC50=71 nM)

Introducing the Chiral Constrained α‐Trifluoromethylalanine in Aib Foldamers to Control, Quantify and Assign the Helical Screw‐Sense**

International audienceOligomers of α-aminoisobutyric acid (Aib) are achiral peptides that adopt 310 helical structures with equal population of left- and right-handed conformers. The screw-sense preference of the helical chain may be controlled by a single chiral residue located at one terminus. 1H and 19F NMR, X-ray crystallography and circular dichroism studies on new Aib oligomers show that the incorporation of a chiral quaternary α-trifluoromethylalanine at their N-terminus induces a reversal of the screw-sense preference of the 310-helix compared to that of a non-fluorinated analogue having an l-α-methyl valine residue. This work demonstrates that, among the many particular properties of introducing a trifluoromethyl group into foldamers, its stereo-electronic properties are of major interest to control the helical screw sense. Its use as an easy-to-handle 19F NMR probe to reliably determine both the magnitude of the screw-sense preference and its sign assignment is also of remarkable interest

Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-\uce\ub1-amanitin conjugates for tumor-targeting

RGD-\uce\ub1-amanitin and isoDGR-\uce\ub1-amanitin conjugates were synthesized by joining integrin ligands to \uce\ub1-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified \uce\ub1V\uce\ub23receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of \uce\ub1V\uce\ub23integrin expression: human glioblastoma U87 (\uce\ub1V\uce\ub23+), human lung carcinoma A549 (\uce\ub1V\uce\ub23\ue2\u88\u92) and breast adenocarcinoma MDA-MB-468 (\uce\ub1V\uce\ub23\ue2\u88\u92). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-\uce\ub1-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than \uce\ub1-amanitin. Apparently, for all these \uce\ub1-amanitin conjugates there is no correlation between the cytotoxicity and the expression of \uce\ub1V\uce\ub23integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-\uce\ub1-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (\uce\ub1V\uce\ub23+, \uce\ub1V\uce\ub25+, \uce\ub1V\uce\ub26\ue2\u88\u92, \uce\ub15\uce\ub21+) and MDA-MB-468 (\uce\ub1V\uce\ub23\ue2\u88\u92, \uce\ub1V\uce\ub25+, \uce\ub1V\uce\ub26+, \uce\ub15\uce\ub21\ue2\u88\u92) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC50was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only \uce\ub1V\uce\ub23, but also \uce\ub1V\uce\ub25, \uce\ub1V\uce\ub26, and \uce\ub15\uce\ub21. These data indicate that in this case the cyclo[DKP-isoDGR]-\uce\ub1-amanitin conjugates are possibly internalized by a process mediated by integrins different from \uce\ub1V\uce\ub23(e.g., \uce\ub1V\uce\ub25)

Optimizing the enzymatic release of MMAE from isoDGR-based small molecule drug conjugate by incorporation of a GPLG-PABC enzymatically cleavable linker

Zambra M, Randelovic I, Talarico F, et al. Optimizing the enzymatic release of MMAE from isoDGR-based small molecule drug conjugate by incorporation of a GPLG-PABC enzymatically cleavable linker. Frontiers in Pharmacology . 2023;14: 1215694.Antibody-Drug Conjugates (ADCs) and Small Molecule-Drug Conjugates (SMDCs) represent successful examples of targeted drug-delivery technologies for overcoming unwanted side effects of conventional chemotherapy in cancer treatment. In both strategies, a cytotoxic payload is connected to the tumor homing moiety through a linker that releases the drug inside or in proximity of the tumor cell, and that represents a key component for the final therapeutic effect of the conjugate. Here, we show that the replacement of the Val-Ala-p-aminobenzyloxycarbamate linker with the Gly-Pro-Leu-Gly-p-aminobenzyloxycarbamate (GPLG-PABC) sequence as enzymatically cleavable linker in the SMDC bearing the cyclo[DKP-isoDGR] alphaVbeta3 integrin ligand as tumor homing moiety and the monomethyl auristatin E (MMAE) as cytotoxic payload led to a 4-fold more potent anti-tumoral effect of the final conjugate on different cancer cell lines. In addition, the synthesized conjugate resulted to be significantly more potent than the free MMAE when tested following the "kiss-and-run" protocol, and the relative potency were clearly consistent with the expression of the alphaVbeta3 integrin receptor in the considered cancer cell lines. In vitro enzymatic cleavage tests showed that the GPLG-PABC linker is cleaved by lysosomal enzymes, and that the released drug is observable already after 15min of incubation. Although additional data are needed to fully characterize the releasing capacity of GPLG-PABC linker, our findings are of therapeutic significance since we are introducing an alternative to other well-established enzymatically sensitive peptide sequences that might be used in the future for generating more efficient and less toxic drug delivery systems. Copyright © 2023 Zambra, Randelovic, Talarico, Borbely, Svajda, Tovari, Mezo, Bodero, Colombo, Arrigoni, Fasola, Gazzola and Piarulli