Peptide-based CE-SELEX enables convenient isolation of aptamers specifically recognizing CD20-expressing cells.

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.

Multimeric RGD-Based Strategies for Selective Drug Delivery to Tumor Tissues.

RGD peptides have received a lot of attention over the two last decades, in particular to improve tumor therapy through the targeting of the αVß3 integrin receptor. This review focuses on the molecular design of multimeric RGD compounds, as well as the design of suitable linkers for drug delivery. Many examples of RGD-drug conjugates have been developed, and we show the importance of RGD constructs to enhance binding affinity to tumor cells, as well as their drug uptake. Further, we also highlight the use of RGD peptides as theranostic systems, promising tools offering dual modality, such as tumor diagnosis and therapy. In conclusion, we address the challenging issues, as well as ongoing and future development, in comparison with large molecules, such as monoclonal antibodies.

Multiplexed Imaging Reveals the Spatial Relationship of the Extracellular Acidity-Targeting pHLIP with Necrosis, Hypoxia, and the Integrin-Targeting cRGD Peptide.

pH (low) insertion peptides (pHLIPs) have been developed for cancer imaging and therapy targeting the acidic extracellular microenvironment. However, the characteristics of intratumoral distribution (ITD) of pHLIPs are not yet fully understood. This study aimed to reveal the details of the ITD of pHLIPs and their spatial relationship with other tumor features of concern. The fluorescent dye-labeled pHLIPs were intravenously administered to subcutaneous xenograft mouse models of U87MG and IGR-OV1 expressing αVß3 integrins (using large necrotic tumors). The αVß3 integrin-targeting Cy5.5-RAFT-c(-RGDfK-)4 was used as a reference. In vivo and ex vivo fluorescence imaging, whole-tumor section imaging, fluorescence microscopy, and multiplexed fluorescence colocalization analysis were performed. The ITD of fluorescent dye-labeled pHLIPs was heterogeneous, having a high degree of colocalization with necrosis. A direct one-to-one comparison of highly magnified images revealed the cellular localization of pHLIP in pyknotic, karyorrhexis, and karyolytic necrotic cells. pHLIP and hypoxia were spatially contiguous but not overlapping cellularly. The hypoxic region was found between the ITDs of pHLIP and the cRGD peptide and the Ki-67 proliferative activity remained detectable in the pHLIP-accumulated regions. The results provide a better understanding of the characteristics of ITD of pHLIPs, leading to new insights into the theranostic applications of pHLIPs.

Multivalent glycocyclopeptides: conjugation methods and biological applications.

Click chemistry was extensively used to decorate synthetic multivalent scaffolds with glycans to mimic the cell surface glycocalyx and to develop applications in glycosciences. Conjugation methods such as oxime ligation, copper(I)-catalyzed alkyne-azide cycloaddition, thiol-ene coupling, squaramide coupling or Lansbury aspartylation proved particularly suitable to achieve this purpose. This review summarizes the synthetic strategies that can be used either in a stepwise manner or in an orthogonal one-pot approach, to conjugate multiple copies of identical or different glycans to cyclopeptide scaffolds (namely multivalent glycocyclopeptides) having different size, valency, geometry and molecular composition. The second part of this review will describe the potential of these structures to interact with various carbohydrate binding proteins or to stimulate immunity against tumor cells.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

2-Hydroxy-4-methoxybenzyl as a Thiol-Protecting Group for Directed-Disulfide Bond Formation.

The chemical synthesis of disulfide-rich peptides such as toxins can be accomplished by using numerous orthogonal cysteine-protecting groups. Herein we report the use of the Hmboff/on protecting group for directed disulfide bond formation. Its combination with classical Trt, Acm, and Mob groups was studied for the synthesis of NMB-1 and phlotoxin-1 toxins highlighting new orthogonal strategies for directed disulfide bond formation.

Ratiometric Luminescence Detection of Copper(I) by a Resonant System Comprising Two Antenna/Lanthanide Pairs.

Selective and sensitive detection of Cu(I) is an ongoing challenge due to its important role in biological systems, for example. Herein, we describe a photoluminescent molecular chemosensor integrating two lanthanide ions (Tb3+ and Eu3+) and respective tryptophan and naphthalene antennas onto a polypeptide backbone. The latter was structurally inspired from copper-regulating biomacromolecules in Gram-negative bacteria and was found to bind Cu+ effectively under pseudobiological conditions (log KCu+ = 9.7 ± 0.2). Ion regulated modulation of lanthanide luminescence in terms of intensity and long, millisecond lifetime offers perspectives in terms of ratiometric and time-gated detection of Cu+. The role of the bound ion in determining the photophysical properties is discussed with the aid of additional model compounds.

Determination of the Rituximab Binding Site to the CD20 Epitope Using SPOT Synthesis and Surface Plasmon Resonance Analyses.

Antibodies not only play a major role in clinical diagnostics and biopharmaceutical analysis but also are a class of drugs that are regularly used to treat numerous diseases. The identification of antibody-epitope binding sites is then of great interest to many emerging medical and bioanalytical applications, particularly to design monoclonal antibodies (mAb) mimics taking advantage of amino acid residues involved in the binding. Among relevant antibodies, the monoclonal antibody rituximab has received significant attention as it is exploited to treat several cancers including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis. The binding of rituximab to the targeted cells occurs via the recognition of the CD20 epitope. A crystallographic study has shown that the binding area, named paratope, is located at the surface of rituximab. Combining the SPOT method and the complementary surface plasmon resonance technique allowed us to detect an extended recognition domain buried in the pocket of the rituximab Fab formed by four ß-sheets. More generally, the present study offers a comprehensive approach to identify antibody-epitope binding sites.

Radiotheranostic Agent 64Cu-cyclam-RAFT-c(-RGDfK-)4 for Management of Peritoneal Metastasis in Ovarian Cancer.

PURPOSE: Ovarian cancer peritoneal metastases (OCPMs) are a pathophysiologically heterogeneous group of tumors that are rarely curable. αVß3 integrin (αVß3) is overexpressed on tumoral neovessels and frequently on ovarian cancer cells. Here, using two clinically relevant αVß3-positive OCPM mouse models, we studied the theranostic potential of an αVß3-specific radiopeptide, 64Cu-cyclam-RAFT-c(-RGDfK-)4 (64Cu-RaftRGD), and its intra- and intertumoral distribution in relation to the tumor microenvironment. EXPERIMENTAL DESIGN: αVß3-expressing peritoneal and subcutaneous models of ovarian carcinoma (IGR-OV1 and NIH:OVCAR-3) were established in nude mice. 64Cu-RaftRGD was administered either intravenously or intraperitoneally. We performed intratumoral distribution (ITD) studies, PET/CT imaging and quantification, biodistribution assay and radiation dosimetry, and therapeutic efficacy and toxicity studies. RESULTS: Intraperitoneal administration was an efficient route for targeting 64Cu-RaftRGD to OCPMs with excellent tumor penetration. Using the fluorescence surrogate, Cy5.5-RaftRGD, in our unique high-resolution multifluorescence analysis, we found that the ITD of 64Cu-RaftRGD was spatially distinct from, but complementary to, that of hypoxia. 64Cu-RaftRGD-based PET enabled clear visualization of multiple OCPM deposits and ascites and biodistribution analysis demonstrated an inverse correlation between tumor uptake and tumor size (1.2-17.2 mm). 64Cu-RaftRGD at a radiotherapeutic dose (148 MBq/0.357 nmol) showed antitumor activities by inhibiting tumor cell proliferation and inducing apoptosis, with negligible toxicity. CONCLUSIONS: Collectively, these results demonstrate the all-in-one potential of 64Cu-RaftRGD for imaging guided radiotherapy of OCPM by targeting both tumoral neovessels and cancerous cells. On the basis of the ITD finding, we propose that pairing αVß3- and hypoxia-targeted radiotherapies could improve therapeutic efficacy by overcoming the heterogeneity of ITD encountered with single-agent treatments.

Using Native Chemical Ligation for Site-Specific Synthesis of Hetero-bis-lanthanide Peptide Conjugates: Application to Ratiometric Visible or Near-Infrared Detection of Zn2.

The interest in ratiometric luminescent probes that detect and quantify a specific analyte is growing. Owing to their special luminescence properties, lanthanide(III) cations offer attractive opportunities for the design of dual-color ratiometric probes. Here, the design principle of hetero-bis-lanthanide peptide conjugates by using native chemical ligation is described for perfect control of the localization of each lanthanide cation within the molecule. Two zinc-responsive probes, r-LZF1Tb|Cs124|Eu and r-LZF1Eu|Cs124|Tb are described on the basis of a zinc finger peptide and two DOTA (DOTA=1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid) complexes of terbium and europium. Both display dual-color ratiometric emission in response to the presence of Zn2+ . By using a screening approach, anthracene was identified for the sensitization of the luminescence of two near-infrared-emitting lanthanides, Yb3+ and Nd3+ . Thus, two novel zinc-responsive hetero-bis-lanthanide probes, r-LZF3Yb|Anthra|Nd and r-LZF3Nd|Anthra|Yb were assembled, the former offering a neat ratiometric response to Zn2+ with emission in the near-infrared around 1000 nm, which is unprecedented.

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.

Impact of Antigen Density on Recognition by Monoclonal Antibodies.

Understanding antigen-antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.

Synthesis and Biological Characterization of Monomeric and Tetrameric RGD-Cryptophycin Conjugates.

The effective delivery of cytotoxic agents to tumor cells is a key challenge in anticancer therapy. Multivalent integrinspecific ligands are considered a promising tool to increase the binding affinity, selectivity, and internalization efficiency of small-molecule drug conjugates. Herein, we report the synthesis and biological evaluation of a multimeric conjugate containing the high-affinity integrin αv ß3 binding ligand RAFT-c(RGDfK)4 , a lysosomally cleavable Val-Cit linker, and cryptophycin-55 glycinate, a potent inhibitor of tubulin polymerization. In vitro cytotoxicity assays verified that the multimeric RGD-cryptophycin conjugate displays improved potency compared to the monomeric analogue in integrin αv ß3 overexpressing tumor cell lines, while significantly reduced activity was observed in the integrin-negative cell line.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

Invited for the cover of this issue is Olivier Renaudet and co-workers at the Université Grenoble Alpes and funded by the European Research Council (CoG "LEGO'" no. 647938). The image illustrates a synthetic chemist playing with supramolecular structures to kill cancer cells by using natural antibodies present in the blood stream. Read the full text of the article at 10.1002/chem.201903327.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

We have developed a fully synthetic and multifunctional antibody-recruiting molecule (ARM) to guide natural antibodies already present in the blood stream against cancer cells without pre-immunization. Our ARM is composed of antibody and tumor binding modules (i.e., ABM and TBM) displaying clustered rhamnose and cyclo-RGD, respectively. By using a stepwise approach, we have first demonstrated the importance of multivalency for efficient recognition with naturel IgM and αv ß3 integrin expressing M21 tumor cell line. Once covalently conjugated by click chemistry, we confirmed by flow cytometry and confocal microscopy that the recognition properties of both the ABM and TBM are conserved, and more importantly, that the resulting ARM promotes the formation of a ternary complex between natural IgM and cancer cells, which is required for the stimulation of the cytotoxic immune response in vivo. Due to the efficiency of the synthetic process, a larger diversity of heterovalent ligands could be easily explored by using the same multivalent approach and could open new perspectives in this field.

Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1.

Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

Synthesis and photophysical studies of a multivalent photoreactive RuII-calix[4]arene complex bearing RGD-containing cyclopentapeptides.

Photoactive ruthenium-based complexes are actively studied for their biological applications as potential theragnostic agents against cancer. One major issue of these inorganic complexes is to penetrate inside cells in order to fulfil their function, either sensing the internal cell environment or exert a photocytotoxic activity. The use of lipophilic ligands allows the corresponding ruthenium complexes to passively diffuse inside cells but limits their structural and photophysical properties. Moreover, this strategy does not provide any cell selectivity. This limitation is also faced by complexes anchored on cell-penetrating peptides. In order to provide a selective cell targeting, we developed a multivalent system composed of a photoreactive ruthenium(II) complex tethered to a calix[4]arene platform bearing multiple RGD-containing cyclopentapeptides. Extensive photophysical and photochemical characterizations of this Ru(II)-calixarene conjugate as well as the study of its photoreactivity in the presence of guanosine monophosphate have been achieved. The results show that the ruthenium complex should be able to perform efficiently its photoinduced cytotoxic activity, once incorporated into targeted cancer cells thanks to the multivalent platform.

Uniform intratumoral distribution of radioactivity produced using two different radioagents, 64Cu-cyclam-RAFT-c(-RGDfK-)4 and 64Cu-ATSM, improves therapeutic efficacy in a small animal tumor model.

BACKGROUND: The present study proposed a new concept for targeted radionuclide therapy (TRT) to improve the intratumoral distribution of radioactivity using two different radiopharmaceuticals. We examined the efficacy of a combination of a tetrameric cyclic Arg-Gly-Asp (cRGD) peptide-based radiopharmaceutical, 64Cu-cyclam-RAFT-c(-RGDfK-)4 (64Cu-RaftRGD, an αVß3 integrin [αVß3] tracer), and 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM, a supposed tracer for hypoxic metabolism) in a small animal tumor model. RESULTS: Mice with subcutaneous αVß3-positive U87MG glioblastoma xenografts were used. The intratumoral distribution of a near-infrared dye, Cy5.5-labeled RAFT-c(-RGDfK-)4 (Cy5.5-RaftRGD), 64Cu-RaftRGD, and 64Cu-ATSM was visualized by fluorescence imaging and autoradiography of the co-injected Cy5.5-RaftRGD with 64Cu-RaftRGD or 64Cu-ATSM at 3 h postinjection. Mice were treated with a single intravenous dose of the vehicle solution (control), 18.5 or 37 MBq of 64Cu-RaftRGD or 64Cu-ATSM, or a combination (18.5 MBq of each agent). The tumor volume, tumor cell proliferation, body weight, survival, and tumor and organ uptake of radiopharmaceuticals were assessed. It was shown that Cy5.5-RaftRGD colocalized with 64Cu-RaftRGD and could be used as a surrogate for the radioactive agent. The intratumoral distribution of Cy5.5-RaftRGD and 64Cu-ATSM was discordant and nearly complementary, indicating a more uniform distribution of radioactivity achievable with the combined use of 64Cu-RaftRGD and 64Cu-ATSM. Neither 64Cu-RaftRGD nor 64Cu-ATSM showed significant effects on tumor growth at 18.5 MBq. The combination of both (18.5 MBq each) showed sustained inhibitory effects against tumor growth and tumor cell proliferation and prolonged the survival of the mice, compared to that by either single agent at 37 MBq. Interestingly, the uptake of the combination by the tumor was higher than that of 64Cu-RaftRGD alone, but lower than that of 64Cu-ATSM alone. The kidneys showed the highest uptake of 64Cu-RaftRGD, whereas the liver exhibited the highest uptake of 64Cu-ATSM. No obvious adverse effects were observed in all treated mice. CONCLUSIONS: The combination of 64Cu-RaftRGD and 64Cu-ATSM achieved an improved antitumor effect owing to the more uniform intratumoral distribution of radioactivity. Thus, combining different radiopharmaceuticals to improve the intratumoral distribution would be a promising concept for more effective and safer TRT.

Design of RGD-ATWLPPR peptide conjugates for the dual targeting of αVß3 integrin and neuropilin-1.

Targeting the tumour microenvironment is a promising strategy to detect and/or treat cancer. The design of selective compounds that co-target several receptors frequently overexpressed in solid tumours may allow a reliable and selective detection of tumours. Here we report the modular synthesis of compounds encompassing ligands of αVß3 integrin and neuropilin-1 that are overexpressed in the tumour microenvironment. These compounds were then evaluated through cellular experiments and imaging of tumours in mice. We observed that the peptide that displays both ligands is more specifically accumulating in the tumours than in controls. Simultaneous interaction with αVß3 integrin and NRP1 induces NRP1 stabilization at the cell membrane surface which is not observed with the co-injection of the controls.

Mercury Trithiolate Binding (HgS3) to a de Novo Designed Cyclic Decapeptide with Three Preoriented Cysteine Side Chains.

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS3 coordination). The model cyclodecapeptide P3C, c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS3 coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P2C cyclodecapeptide validated previously as a model for d10 metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP3C complex with a HgS3 coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and 199Hg NMR. Hg LIII-edge extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the Hg(II) coordination environment is T-shaped with two short Hg-S distances at 2.45 Å and one longer distance at 2.60 Å. The solution structure of the HgP3C complex was refined based on 1H-1H NMR constraints and EXAFS results. The cyclic peptide scaffold has a rectangular shape with the three binding cysteine side chains pointing toward Hg(II). The HgP3CH complex has a p Ka of 4.3, indicating that the HgS3 coordination mode is stable over a large range of pH. This low p Ka value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P3C.