Amine-Carbamate Self-Immolative Spacers Counterintuitively Release 3° Alcohol at Much Faster Rates than 1° Alcohol Payloads.

Self-immolative (SI) spacers are degradable chemical connectors widely used in prodrugs and drug conjugates to release pharmaceutical ingredients in response to specific stimuli. Amine-carbamate SI spacers are particularly versatile, as they have been used to release different hydroxy cargos, ranging from 2° and 3° alcohols to phenols and oximes. In this work, we describe the ability of three amine-carbamate SI spacers to release three structurally similar imidazoquinoline payloads, bearing either a 1°, a 2° or a 3° alcohol as the leaving group. While the spacers showed comparable efficacy at releasing the 2° and 3° alcohols, the liberation of the 1° alcohol was much slower, unveiling a counterintuitive trend in nucleophilic acyl substitutions. The release of the 1° alcohol payload was only possible using a SI spacer bearing a pyrrolidine ring and a tertiary amine handle, which opens the way to future applications in drug delivery systems.

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.

Visible Light-Promoted ß-Functionalization of Carbonyl Compounds in the Presence of Organic Dyes.

Herein, we investigate the use of organic photocatalysts in the visible light-promoted ß-functionalization of carbonyl compounds. In particular, we studied the addition of aliphatic aldehydes to α,ß-unsaturated compounds (ß-Michael addition), and the reaction of cyclic ketones with either ketones (ß-aldol condensation) or imines (ß-Mannich reaction). Among the dyes tested, donor-acceptor cyanoarenes gave the best results, promoting the transformations of interest in moderate to good yields. The reaction scope was investigated on substrates with different steric and electronic properties. Fluorescence quenching analysis (Stern-Volmer experiments) led us to propose for these reactions a reductive quenching mechanism involving a transient 5πe- activation mode.

RGD Cyclopeptide Equipped with a Lysine-Engaging Salicylaldehyde Showing Enhanced Integrin Affinity and Cell Detachment Potency.

Salicylaldehyde (SA) derivatives are emerging as useful fragments to obtain reversible-covalent inhibitors interacting with the lysine residues of the target protein. Here the SA installation at the C terminus of an integrin-binding cyclopeptide, leading to enhanced ligand affinity for the receptor as well as stronger biological activity in cultured glioblastoma cells is reported.

Advanced Pyrrolidine-Carbamate Self-Immolative Spacer with Tertiary Amine Handle Induces Superfast Cyclative Drug Release.

Amine-carbamate self-immolative (SI) spacers represent practical and versatile tools in targeted prodrugs, but their slow degradation mechanism limits drug activation at the site of disease. We engineered a pyrrolidine-carbamate SI spacer with a tertiary amine handle which strongly accelerates the spacer cyclization to give a bicyclic urea and the free hydroxy groups of either cytotoxic (Camptothecin) or immunostimulatory (Resiquimod) drugs. In silico conformational analysis and pKa calculations suggest a plausible mechanism for the superior efficacy of the advanced SI spacer compared to state-of-art analogues.

Development and Biochemical Characterization of Self-Immolative Linker Containing GnRH-III-Drug Conjugates.

The human gonadotropin releasing hormone (GnRH-I) and its sea lamprey analogue GnRH-III specifically bind to GnRH receptors on cancer cells and can be used as targeting moieties for targeted tumor therapy. Considering that the selective release of drugs in cancer cells is of high relevance, we were encouraged to develop cleavable, self-immolative GnRH-III-drug conjugates which consist of a p-aminobenzyloxycarbonlyl (PABC) spacer between a cathepsin B-cleavable dipeptide (Val-Ala, Val-Cit) and the classical anticancer drugs daunorubicin (Dau) and paclitaxel (PTX). Alongside these compounds, non-cleavable GnRH-III-drug conjugates were also synthesized, and all compounds were analyzed for their antiproliferative activity. The cleavable GnRH-III bioconjugates revealed a growth inhibitory effect on GnRH receptor-expressing A2780 ovarian cancer cells, while their activity was reduced on Panc-1 pancreatic cancer cells exhibiting a lower GnRH receptor level. Moreover, the antiproliferative activity of the non-cleavable counterparts was strongly reduced. Additionally, the efficient cleavage of the Val-Ala linker and the subsequent release of the drugs could be verified by lysosomal degradation studies, while radioligand binding studies ensured that the GnRH-III-drug conjugates bound to the GnRH receptor with high affinity. Our results underline the high value of GnRH-III-based homing devices and the application of cathepsin B-cleavable linker systems for the development of small molecule drug conjugates (SMDCs).

A trifunctional self-immolative spacer enables drug release with two non-sequential enzymatic cleavages.

Cyclative cleavage of an amine-carbamate self-immolative spacer to deliver a hydroxyl cargo was inhibited by spacer derivatisation with a phosphate monoester handle. This trifunctional spacer was installed in a model anticancer prodrug that showed fast drug release only when incubated with both a protease and a phosphatase enzyme.

Multimeric Presentation of RGD Peptidomimetics Enhances Integrin Binding and Tumor Cell Uptake.

The use of multimeric ligands is considered as a promising strategy to improve tumor targeting for diagnosis and therapy. Herein, tetrameric RGD (Arg-Gly-Asp) peptidomimetics were designed to target αv ß3 integrin-expressing tumor cells. These compounds were prepared by an oxime chemoselective assembly of cyclo(DKP-RGD) ligands and a cyclodecapeptide scaffold, which allows a tetrameric presentation. The resulting tetrameric RGD peptidomimetics were shown to improve αv ß3 integrin binding compared with the monomeric form. Interestingly, these compounds were also able to enhance tumor cell endocytosis in the same way as tetrameric RGD peptides. Altogether, the results show the potential of the tetrameric cyclo(DKP-RGD) ligands for in vivo imaging and drug delivery.

Fast Cyclization of a Proline-Derived Self-Immolative Spacer Improves the Efficacy of Carbamate Prodrugs.

Self-immolative (SI) spacers are sophisticated chemical constructs designed for molecular delivery or material degradation. We describe herein a (S)-2-(aminomethyl)pyrrolidine SI spacer that is able to release different types of anticancer drugs (possessing either a phenolic or secondary and tertiary hydroxyl groups) through a fast cyclization mechanism involving carbamate cleavage. The high efficiency of drug release obtained with this spacer was found to be beneficial for the in vitro cytotoxic activity of protease-sensitive prodrugs, compared with a commonly used spacer of the same class. These findings expand the repertoire of degradation machineries and are instrumental for the future development of highly efficient delivery platforms.

A dimeric bicyclic RGD ligand displays enhanced integrin binding affinity and strong biological effects on U-373 MG glioblastoma cells.

A C2-symmetric bicyclic peptide bearing two RGD motifs was developed as a dimeric ligand, and it displayed enhanced inhibition of ECM protein binding to purified integrin receptors as compared to monomeric RGD analogues. Moreover, the dimeric bicyclic ligand induced cell detachment and inhibited FAK phosphorylation in U-373 MG glioblastoma cells.

Innovative Linker Strategies for Tumor-Targeted Drug Conjugates.

The covalent conjugation of potent cytotoxic agents to either macromolecular carriers or small molecules represents a well-known approach to increase the therapeutic index of these drugs, thus improving treatment efficacy and minimizing side effects. In general, cytotoxic activity is displayed only upon cleavage of a specific chemical bond (linker) that connects the drug to the carrier. The perfect balance between the linker stability and its selective cleavage represents the key for success in these therapeutic approaches and the chemical toolbox to reach this goal is continuously expanding. In this Review article, we highlight recent advances on the different modalities to promote the selective release of cytotoxic agents, either by exploiting specific hallmarks of the tumor microenvironment (e.g. pH, enzyme expression) or by the application of external triggers (e.g. light and bioorthogonal reactions).

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

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 αvß3, 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 αvß3, 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 αvß3 expression level was observed, which is presumably due to a non-integrin-mediated uptake. This reveals the complexity of effective and selective αvß3 integrin-mediated drug delivery.

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.

ß-Glucuronidase triggers extracellular MMAE release from an integrin-targeted conjugate.

A non-internalizing αvß3 integrin ligand was conjugated to the anticancer drug MMAE through a ß-glucuronidase-responsive linker. In the presence of ß-glucuronidase, only the conjugate bearing a PEG4 spacer inhibited the proliferation of integrin-expressing cancer cells at low nanomolar concentrations, indicating important structural requirements for the efficacy of these therapeutics.

Synthesis and Biological Evaluation of RGD and isoDGR-Monomethyl Auristatin Conjugates Targeting Integrin αV ß3.

This work reports the synthesis of a series of small-molecule-drug conjugates containing the αV ß3 -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 ß3 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 ß3 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.

The Importance of Detail: How Differences in Ligand Structures Determine Distinct Functional Responses in Integrin αv ß3.

Ligand-based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand-based modulation of integrin functions. Inhibitors of integrin αv ß3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg-Gly-Asp (RGD) or isoAsp-Gly-Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo-RGDf(NMe)V (Cilengitide), cyclo[DKP3-RGD], cyclo[DKP3-isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand-controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site.

Neutrophil Elastase Promotes Linker Cleavage and Paclitaxel Release from an Integrin-Targeted Conjugate.

This work takes advantage of one of the hallmarks of cancer, that is, the presence of tumor infiltrating cells of the immune system and leukocyte-secreted enzymes, to promote the activation of an anticancer drug at the tumor site. The peptidomimetic integrin ligand cyclo(DKP-RGD) was found to accumulate on the surface of αv ß3 integrin-expressing human renal cell carcinoma 786-O cells. The ligand was conjugated to the anticancer drug paclitaxel through a Asn-Pro-Val (NPV) tripeptide linker, which is a substrate of neutrophil-secreted elastase. In vitro linker cleavage assays and cell antiproliferative experiments demonstrate the efficacy of this tumor-targeting conjugate, opening the way to potential therapeutic applications.

Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting.

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified αVß3 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 αVß3 integrin expression: human glioblastoma U87 (αVß3+), human lung carcinoma A549 (αVß3-) and breast adenocarcinoma MDA-MB-468 (αVß3-). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of αVß3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-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 (αVß3+, αVß5+, αVß6-, α5ß1+) and MDA-MB-468 (αVß3-, αVß5+, αVß6+, α5ß1-) 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 αVß3, but also αVß5, αVß6, and α5ß1. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from αVß3 (e.g., αVß5).

Investigating the Interaction of Cyclic RGD Peptidomimetics with αVß6 Integrin by Biochemical and Molecular Docking Studies.

The interaction of a small library of cyclic RGD (Arg-Gly-Asp) peptidomimetics with αVß6 integrin has been investigated by means of competitive solid phase binding assays to the isolated receptor and docking calculations in the crystal structure of the αVß6 binding site. To this aim, a rigid receptor-flexible ligand docking protocol has been set up and then applied to predict the binding mode of the cyclic RGD peptidomimetics to αVß6 integrin. Although the RGD interaction with αVß6 recapitulates the RGD binding mode observed in αVß3, differences between the integrin binding pockets can strongly affect the ligand binding ability. In general, the peptidomimetics exhibited IC50 values for integrin αVß6 (i.e., the concentration of compound required for 50% inhibition of biotinylated fibronectin binding to isolated αVß6 integrin) in the nanomolar range (77-345 nM), about 10-100 times higher than those for the related αVß3 receptor, with a single notable ligand displaying a low nanomolar IC50 value (2.3 nM). Insights from the properties of the binding pocket combined with the analysis of the docking poses provided a rationale for ligand recognition and selectivity.

Multivalency Increases the Binding Strength of RGD Peptidomimetic-Paclitaxel Conjugates to Integrin αV ß3.

This work reports the synthesis of three multimeric RGD peptidomimetic-paclitaxel conjugates featuring a number of αV ß3 integrin ligands ranging from 2 to 4. These constructs were assembled by conjugation of the integrin αV ß3 ligand cyclo[DKP-RGD]-CH2 NH2 with paclitaxel via a 2'-carbamate with a self-immolative spacer, the lysosomally cleavable Val-Ala dipeptide linker, a multimeric scaffold, a triazole linkage, and finally a PEG spacer. Two monomeric conjugates were also synthesized as reference compounds. Remarkably, the new multimeric conjugates showed a binding affinity for the purified integrin αV ß3 receptor that increased with the number of integrin ligands (reaching a minimum IC50 value of 1.2 nm for the trimeric), thus demonstrating that multivalency is an effective strategy to strengthen the ligand-target interactions.