Synthesis and Biological Characterization of Monomeric and Tetrameric RGD-Cryptophycin Conjugates.
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ABSTRACT:
SUBMITTER: Borbely A
PROVIDER: S-EPMC7064988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin ?v??, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)?cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin ?v?? expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.
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Project description:Most anticancer agents are hydrophobic and can easily penetrate the tumor cell membrane by passive diffusion. This may impede the development of highly effective and tumor-selective treatment options. A hydrophilic ?-glucuronidase-cleavable linker was used to connect the highly potent antimitotic agent cryptophycin-55 glycinate with the ?v?3 integrin ligand c(RGDfK). Incorporation of the self-immolative linker containing glucuronic acid results in decreased cytotoxicity compared to the free payload, suggesting that hydrophilic sugar linkers can preclude passive cellular uptake. In vitro drug-release studies and cytotoxicity assays demonstrated the potential of this small molecule-drug conjugate, providing guidance for the development of therapeutics containing hydrophobic anticancer drugs.
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Project description: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 ?v?3, 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 ?v?3, 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 ?v?3 expression level was observed, which is presumably due to a non-integrin-mediated uptake. This reveals the complexity of effective and selective ?v?3 integrin-mediated drug delivery.
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Project description:RGD-?-amanitin and isoDGR-?-amanitin conjugates were synthesized by joining integrin ligands to ?-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified ?V?3 receptor, 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 ?V?3 integrin expression: human glioblastoma U87 (?V?3+), human lung carcinoma A549 (?V?3-) and breast adenocarcinoma MDA-MB-468 (?V?3-). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-?-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than ?-amanitin. Apparently, for all these ?-amanitin conjugates there is no correlation between the cytotoxicity and the expression of ?V?3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-?-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 (?V?3+, ?V?5+, ?V?6-, ?5?1+) and MDA-MB-468 (?V?3-, ?V?5+, ?V?6+, ?5?1-) 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 IC50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only ?V?3, but also ?V?5, ?V?6, and ?5?1. These data indicate that in this case the cyclo[DKP-isoDGR]-?-amanitin conjugates are possibly internalized by a process mediated by integrins different from ?V?3 (e.g., ?V?5).
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Project description:Treatment of CuCl with 1 equiv of the in situ prepared N-mesityl-substituted diamidocarbene 6-MesDAC produced a mixture of the dimeric and trimeric copper complexes [(6-MesDAC)CuCl]2 (1) and [(6-MesDAC)2(CuCl)3] (2). Combining CuCl with isolated, free 6-MesDAC in 1:1 and 3:2 ratios gave just 1 and 2, respectively, while increasing the ratio to >5:1 allowed the isolation of small amounts of the tetrameric copper complex [(6-MesDAC)2(CuCl)4] (3). Efforts to bring about metathesis reactions of 1 with MO(t)Bu (M = Li, Na, K) proved successful only for M = Li to afford the spectroscopically characterized ate product [(6-MesDAC)CuCl·LiO(t)Bu·2THF] (5). Attempts to crystallize this species instead gave a 1:1 mixture of 1 and the monomer [(6-MesDAC)CuCl] (6). The X-ray structures of 1-3 and 1 + 6, along with the cation [Cu(6-MesDAC)2](+) (4), have been determined.
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Project description:This work reports the synthesis of a series of small-molecule-drug conjugates containing the αV β3 -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 the cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide-alkyne cycloaddition as the key step. The conjugates were tested for their binding affinity for the isolated αv β3 receptor and were shown to retain nanomolar IC50 values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with αv β3 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 IC50 values in cell viability assays with both cancer cell lines tested (U87: 11.50±0.13 nm; M21: 6.94±0.09 nm) and is therefore a promising candidate for in vivo experiments.
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