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.

A Tight Contact: The Expanding Application of Salicylaldehydes in Lysine-Targeting Covalent Drugs.

The installation of aldehydes into synthetic protein ligands is an efficient strategy to engage protein lysine residues in remarkably stable imine bonds and augment the compound affinity and selectivity for their biological targets. The high frequency of lysine residues in proteins and the reversibility of the covalent ligand-protein bond support the application of aldehyde-bearing ligands, holding promises for their future use as drugs. This review highlights the increasing exploitation of salicylaldehyde modules in various classes of protein binders, aimed at the reversible-covalent engagement of lysine residues.

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.

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.

Targeted Small-Molecule Conjugates: The Future is Now.

In recent years, novel classes of small hetero-bifunctional compounds have been developed as tools for the treatment of cancer and other diseases through different approaches, from radio/chemotherapy and imaging to immunotherapy and protein degradation. Within this growing research area, progresses in ligand-screening procedures and hit-to-lead optimizations, together with advances in drug-release technologies, are paving the way to the future treatment of a broad range of pathologies with small targeted therapeutics.

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).

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.

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.

Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues.

The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small-molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1 H NMR techniques. 2-Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small-molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand-binding site and could be abrogated by quenching with a molar excess of hydroxylamine.

Chemically Defined Antibody- and Small Molecule-Drug Conjugates for in Vivo Tumor Targeting Applications: A Comparative Analysis.

We present the first direct comparative evaluation of an antibody-drug conjugate and of a small molecule-drug conjugate for cancer therapy, using chemically defined products which bind with high-affinity to carbonic anhydrase IX, a marker of tumor hypoxia and of renal cell carcinoma.

A non-internalizing antibody-drug conjugate based on an anthracycline payload displays potent therapeutic activity in vivo.

Antibody-drug conjugates are generally believed to crucially rely on internalization into cancer cells for therapeutic activity. Here, we show that a non-internalizing antibody-drug conjugate, based on the F16 antibody specific to the alternatively spliced A1 domain of tenascin-C, mediates a potent therapeutic activity when equipped with the anthracycline PNU159682. The peptide linker, connecting the F16 antibody in IgG format at a specific cysteine residue to the drug, was stable in serum but could be efficiently cleaved in the subendothelial extracellular matrix by proteases released by the dying tumor cells. The results indicate that there may be a broader potential applicability of non-internalizing antibody-drug conjugates for cancer therapy than what had previously been assumed.

Impact of a Central Scaffold on the Binding Affinity of Fragment Pairs Isolated from DNA-Encoded Self-Assembling Chemical Libraries.

The screening of encoded self-assembling chemical libraries allows the identification of fragment pairs that bind to adjacent pockets on target proteins of interest. For practical applications, it is necessary to link these ligand pairs into discrete organic molecules, devoid of any nucleic acid component. Here we describe the discovery of a synergistic binding pair for acid alpha-1 glycoprotein and a chemical strategy for the identification of optimal linkers, connecting the two fragments. The procedure yielded a set of small organic ligands, the best of which exhibited a dissociation constant of 9.9 nm, as measured in solution by fluorescence polarization.

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.