Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging.

Biomolecules labeled with positron-emitting radionuclides like fluorine-18 or radiometals like copper-64 and zirconium-89 are increasingly employed in nuclear medicine for diagnosis purposes. Given the fragility and complexity of these compounds, their labeling requires mild conditions. Besides, it is essential to develop methods inducing minimal modification of the tertiary structure, as it is fundamental for the biological activity of such complex entities. Given these requirements, disulfide rebridging represents a promising possibility since it allows protein modification as well as conservation of the tertiary structure. In this context, we have developed an original radiofluorinated dibromopyridazine dione prosthetic group for labeling of disulfide-containing biomolecules via rebridging. We employed it to radiolabel octreotide, a somatostatin analogue, and to radiolabel fragment antigen binding (Fab) targeting programmed death-ligand 1 (PD-L1), whose properties were then evaluated in vitro and in vivo by positron emission tomography (PET) imaging. We next extended our strategy to the radiolabeling of cetuximab, a monoclonal antibody, with various radiometals commonly used in PET imaging (zirconium-89, copper-64) by developing various rebridging molecules bearing the appropriate chelators. The stabilities of the radiolabeled antibody conjugates were assessed in biological conditions.

NRP1 inhibition modulates radiosensitivity of medulloblastoma by targeting cancer stem cells.

BACKGROUND: Medulloblastoma (MB) is the most common pediatric malignant brain tumor. Despite current therapies, the morbidity and recurrent risk remains significant. Neuropilin-1 receptor (NRP1) has been implicated in the tumor progression of MB. Our recent study showed that NRP1 inhibition stimulated MB stem cells differentiation. Consequently, we hypothesized that targeting NRP1 in medulloblastoma could improve current treatments. METHODS: NRP1 inhibition with a novel peptidomimetic agent, MR438, was evaluated with radiotherapy (RT) in MB models (DAOY, D283-Med and D341-Med) in vitro on cancer stem-like cells as well as in vivo on heterotopic and orthotopic xenografts. RESULTS: We show that NRP1 inhibition by MR438 radiosensitizes MB stem-like cells in vitro. In heterotopic DAOY models, MR438 improves RT efficacy as measured by tumor growth and mouse survival. In addition, clonogenic assays after tumor dissociation showed a significant reduction in cancer stem cells with the combination treatment. In the same way, a benefit of the combined therapy was observed in the orthotopic model only for a low cumulative irradiation dose of 10 Gy but not for 20 Gy. CONCLUSIONS: Finally, our results demonstrated that targeting NRP1 with MR438 could be a potential new strategy and could limit MB progression by decreasing the stem cell number while reducing the radiation dose.

Impact of Radiolabeling Strategies on the Pharmacokinetics and Distribution of an Anti-PD-L1 PET Ligand.

Molecular imaging with PET offers an alternative method to quantify programmed-death-ligand 1 (PD-L1) to accurately select patients for immunotherapies. More and more clinical and preclinical trials involve radiolabeling of antibody fragments for their desirably fast clearance and high tumor penetration. As the radiolabeling strategy can significantly impact pharmacokinetics and biodistribution, we explored in this work a site-specific radiofluorination strategy on an anti-PD-L1 fragment antigen-binding (Fab) and compared the pharmacokinetic and biodistribution properties with the same Fab labeled using stochastic radiolabeling chemistry. We applied an enzymatic bioconjugation mediated by a variant of the lipoic acid ligase (LplA) that promotes the formation of an amide bond between a short peptide cloned onto the C terminus of the Fab. A synthetic analogue of the enzyme natural substrate, lipoic acid, was radiolabeled with fluorine-18 for site-specific conjugation by LplA. We compared the biodistribution of the site-specifically labeled Fab with a stochastically labeled Fab on lysine side chains in tumor-bearing mice. The two methods of fluorination demonstrate a comparable whole-body biodistribution. The 89Zr-labeled Fab had different biodistribution compared to either 18F-labeled Fab. We attribute the difference to [89Zr] metabolism. Fab-LAP-[18F]FPyOctA therefore reflects better the true pharmacokinetic profile of the Fab.

[18 F]FPyZIDE: A versatile prosthetic reagent for the fluorine-18 radiolabeling of biologics via copper-catalyzed or strain-promoted alkyne-azide cycloadditions.

Methods for the radiolabeling of biologics with fluorine-18 have been of interest for several decades. A common approach consists in the preparation of a prosthetic reagent, a small molecule bearing a fluorine-18 that is conjugated with the macromolecule to an appropriate function. Click chemistry, and more particularly cycloadditions, is an interesting approach to radiolabel molecules thanks to mild reaction conditions, high yields, low by-products formation, and strong orthogonality. Moreover, the chemical functions involved in the cycloaddition reaction are stable in the drastic radiofluorination conditions, thus allowing a simple radiosynthetic route to prepare the prosthetic reagent. We report herein the radiosynthesis of 18 F-FPyZIDE, a pyridine-based azide-bearing prosthetic reagent. We exemplified its conjugation via copper-catalyzed cycloaddition (CuAAC) and strain-promoted cycloaddition (SPAAC) with several terminal alkyne or strained alkyne model compounds.

Overt language production plays a key role in the Hebb repetition effect.

When asked to recall verbatim a short list of items, performance is very limited. However, if the list of items is repeated across trials, recall performance improves. This phenomenon, known as the Hebb repetition effect (Hebb, 1961; Brain Mechanisms and Learning: A Symposium, pp. 37-51), is considered a laboratory analogue of language learning. In effect, learning a new word implies the maintenance of a series of smaller units, such as phonemes or syllables, in the correct order for a short amount of time before producing them. The sequence of smaller units is typically presented more than once. In the present study, we investigated the role of overt language production in language learning by manipulating recall direction. If the learning of a repeated list of items relies on overt language production processes, changing list production order by manipulating recall direction should impact the learning of the list. In Experiment 1, one list was repeated every third trial, and recall direction of the repeated list changed on the ninth repetition. In Experiment 1a, the repeated list changed from a forward to a backward order recall, where participants had to recall the items in reverse presentation order. In Experiment 1b, the repeated list changed from a backward to a forward order recall. Results showed a cost in recall performance for the repeated list when recall direction switched from forward to backward recall, whereas it was unaffected by the change from backward to forward recall. In Experiment 2, we increased the number of trials before introducing the change from a backward to a forward order recall. Results showed a decrement in recall performance for the repeated list following the change in recall direction, suggesting that language production processes play a role in the Hebb repetition effect.

Carbohydrate-based peptidomimetics targeting neuropilin-1: Synthesis, molecular docking study and in vitro biological activities.

Neuropilin-1 (NRP-1), a transmembrane glycoprotein acting as a co-receptor of VEGF-A, is expressed by cancer and angiogenic endothelial cells and is involved in the angiogenesis process. Taking advantage of functionalities and stereodiversities of sugar derivatives, the design and the synthesis of carbohydrate based peptidomimetics are here described. One of these compounds (56) demonstrated inhibition of VEGF-A165 binding to NRP-1 (IC50=39µM) and specificity for NRP-1 over VEGF-R2. Biological evaluations were performed on human umbilical vein endothelial cells (HUVECs) through activation of downstream proteins (AKT and ERK phosphorylation), viability/proliferation assays and in vitro measurements of anti-angiogenic abilities.

Novel [18F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.

BACKGROUND: Prosthetic approach for the radiolabeling of biologics with fluorine-18 is a robust strategy and has been employed for many years. It requires fast, biocompatible and selective reactions suited to these fragile molecules. Michael addition of a nucleophilic thiol moiety on α,ß-unsaturated carbonyl entities is an interesting compromise between simplicity of preparation of the prosthetic reagent and control of the selectivity of the addition. The α,ß-unsaturated carbonyl entity of the biologic can easily be generated by addition of a maleimide function using adequate heterobifunctional linkers or generated by selective modification of a cysteine residue leading to a dehydroalanine moiety. We report here the design, synthesis and radiosynthesis of a new fluoropyridine-based thiol [18F]FPySH and its conjugation via Michael addition on model dehydroalanine- or maleimide-containing biologics. RESULTS: The preparation of cold reference and labeling precursor of [18F]FPySH was achieved and its radiosynthesis was fully automated, enabling production of the thiol prosthetic group with a 7 ± 2.1% radiochemical yield after two steps. The conjugation of [18F]FPySH to two model Dha-containing molecules was then carried out in reducing conditions, yielding the corresponding adducts in 30-45 min reaction time. Furthermore, [18F]FPySH was employed to radiolabel the maleimide-modified c(RGDfK) peptide, affording the radiofluorinated analogue in 15 min. CONCLUSION: We have developed an original [18F]-labeled thiol for site-selective conjugation and radiolabeling of Dha or maleimide-containing biomolecules of interest. Labeling of three model compounds was successfully carried out and gave the expected radiofluorinated adducts in less than 45 min, thus compatible with fluorine-18 half-life.

Original synthesis of radiolabeling precursors for batch and on resin one-step/late-stage radiofluorination of peptides.

Radiolabeling of peptides with fluorine-18 is hurdled by their chemical sensitivity and complicated processes. Original triflyl-pyridine intermediates afforded ammonium precursors that were radiolabeled at low temperature. From that study, a generic tag has been designed to allow a simple one-step/late-stage radiolabelling of peptides. The strategy has been transposed to an automated "on-resin" radiolabelling.

New fluorine-18 pretargeting PET imaging by bioorthogonal chlorosydnone-cycloalkyne click reaction.

We report the first pretargeting in vivo study using the Strain-Promoted Sydnone-Alkyne Cycloaadition (SPSAC) reaction. The injection of a fluorine-18 labeled cyclooctyne three days after cetuximab bearing chlorosydnone moieties allowed a significant detection of the tumor by PET imaging suggesting an efficient click reaction inside the tumoral site. With a kinetic constant superior to 300 M-1 s-1, the SPSAC reaction might be an interesting tool, in addition to tetrazine-cyclooctene ligation, for in vivo chemistry.

Stimulation of medulloblastoma stem cells differentiation by a peptidomimetic targeting neuropilin-1.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite the progress of new treatments, the risk of recurrence, morbidity, and death remains important. The neuropilin-1 (NRP-1) receptor has recently been implicated in tumor progression of MB, which seems to play an important role in the phenotype of cancer stem cells. Targeting this receptor appears as an interesting strategy to promote MB stem cells differentiation. Cancer stem-like cells of 3 MB cell lines (DAOY, D283-Med and D341-Med), classified in the more pejorative molecular subgroups, were obtained by in vitro enrichment. These models were characterized by an increase of NRP-1 and cancer stem cell markers (CD15, CD133 and Sox2), meanwhile a decrease of the differentiated cell marker Neurofilament-M (NF-M) was observed. Our previous work investigated potential innovative peptidomimetics that specifically target NRP-1 and showed that MR438 had a good affinity for NRP-1. This small molecule decreased the self-renewal capacity of MB stem cells for the 3 cell lines and reduced the invasive ability of DAOY and D283 stem cells while NRP-1 expression and cancer stem cell markers decreased at the same time. Possible molecular mechanisms were explored and showed that the activation of PI3K/AKT and MAPK pathways significantly decreased for DAOY cells after treatment. Finally, our results highlighted that targeting NRP-1 with MR438 could be a potential new strategy to differentiate MB stem cells and could limit medulloblastoma progression.

Spiro sugar-isoxazolidine scaffold as useful polyfunctional building block for peptidomimetics design.

Spiro sugar-isoxazolidines obtained by 1,3-dipolar cycloaddition of activated exo-glycals and nitrones were efficiently functionalized at two sites, i.e. C-4 and C-7, with arginine, arginine mimetics and guanidylated appendages. Two bicyclic sugar derivatives differing by the configuration at C-7 were chosen as model compounds. The small library of peptidomimetics was evaluated toward inhibition of VEGF-A165/neuropilin-1 binding. Unexpected cleavage of C3-C4 bond of isoxazolidine moiety was observed during hydrogenolysis and opened thus a new way toward hemiketal structures which could also find interesting applications as less constrained scaffold.