Radiolabeled peptides and their expanding role in clinical imaging and targeted cancer therapy.

There is an expanding body of evidence showing that synthetic peptides in combination with radioactive isotopes can be utilized for medical purposes. This area is of particular interest in oncology where applications in diagnosis and therapy are at different stages of development. We review the contributions in this area by the group originally founded by Carlo Pedone in Naples many years ago. We highlight the work of this group in the context of other developments in this area, focusing on three biologically relevant receptor systems: somatostatin, gastrin-releasing peptide, and cholecystokinin-2/gastrin receptors. We focus on key milestones, state of the art, and challenges in this area of research as well as the current and future outlook for expanding clinical applications.

Fluorescence Studies: A9 Peptide, Functionalized with a Fluorogenic Probe, Interacts with Its Receptor Model HER2-DIVMP.

A recently developed synthetic protocol allowed for the functionalization of the active peptide A9 with a fluorogenic probe, which is useful for studying biomolecular interactions. Essentially, a nucleophilic attack on a halo-substituted benzofurazan is selectively performed by a cysteine sulfhydryl group. The process is assisted by the basic catalysis of activated zeolites (4 Å molecular sieves) and promoted by microwave irradiation. Fluorescence studies revealed that a donor-acceptor pair within the peptide sequence was introduced, thus allowing a deeper investigation on the interaction process between the peptide ligand and its receptor fragment. The obtained results allowed us to come full circle for all the currently understood structural determinants that were found to be involved in the binding process.

Microwave Heating Promotes the S-Alkylation of Aziridine Catalyzed by Molecular Sieves: A Post-Synthetic Approach to Lanthionine-Containing Peptides.

Aziridine derivatives involved in nucleophilic ring-opening reactions have attracted great interest, since they allow the preparation of biologically active molecules. A chemoselective and mild procedure to convert a peptide cysteine residue into lanthionine via S-alkylation on aziridine substrates is presented in this paper. The procedure relies on a post-synthetic protocol promoted by molecular sieves to prepare lanthionine-containing peptides and is assisted by microwave irradiation. In addition, it represents a valuable alternative to the stepwise approach, in which the lanthionine precursor is incorporated into peptides as a building block.

Definition of miRNA Signatures of Nodal Metastasis in LCa: miR-449a Targets Notch Genes and Suppresses Cell Migration and Invasion.

Laryngeal cancer (LCa), a neoplasm of the head and neck region, is a leading cause of death worldwide. Surgical intervention remains the mainstay of LCa treatment, but a crucial point is represented by the possible nodal involvement. Therefore, it is urgently needed to develop biomarkers and therapeutic tools able to drive treatment approaches for LCa. In this study, we investigated deregulated microRNAs (miRNAs) in tissues from LCa patients with either lymph node metastases (N+) or not (N-). miRNA expression profiling was performed by a comprehensive PCR array and subsequent validation by RT-qPCR. Results showed a significant decrease of miR-449a expression in N+ compared to N- patients, and miR-133b down-modulation in LCa tissues compared to paired normal ones. Receiver operating characteristic (ROC) curve analysis revealed the potential diagnostic power of miR-133b for LCa detection. According to the validation results, we selected miR-449a for further in vitro studies. Ectopic miR-449a expression in the LCa cell line Hep-2 inhibited invasion and motility in vitro, slowed cell proliferation, and induced the downregulation of Notch1 and Notch2 as direct targets of miR-449a. Collectively, this study provides new promising biomarkers for LCa diagnosis and a new opportunity to use miR-449a for the treatment of nodal metastases in LCa patients.

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study.

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM-SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM-SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM-SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM-SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM-SAM interactions.

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target.

The binding process of A9 peptide toward HER2-DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2-DIVMP, multiple ligand-based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.

Lanthionine Peptides by S-Alkylation with Substituted Cyclic Sulfamidates Promoted by Activated Molecular Sieves: Effects of the Sulfamidate Structure on the Yield.

A green and efficient method for preparing lanthionine peptides by a highly chemoselective and stereochemically controlled procedure is presented. It involves an S-alkylation reaction, promoted by activated molecular sieves, on chiral cyclic sulfamidates, both N-protected and unprotected. Of note, the reaction yield was high also for cyclic sulfamidates bearing a free amine group, while other strategies failed to achieve a ring-opening nucleophilic reaction with N-unprotected substrates. To prove the feasibility of the procedure, the synthesis of a thioether ring B mimetic of the natural lantibiotic haloduracin ß was performed.

Design and analysis of EphA2-SAM peptide ligands: A multi-disciplinary screening approach.

EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM.

A Late-Stage Synthetic Approach to Lanthionine-Containing Peptides via S-Alkylation on Cyclic Sulfamidates Promoted by Molecular Sieves.

A one-pot, high-yield procedure for synthesizing lanthionine-containing peptides was developed. It relies on the S-alkylation of cysteine-containing peptides with chiral cyclic sulfamidates. The key feature of this approach is the use of mild reaction conditions (only activated molecular sieves are employed as the catalyst), leading to good chemoselectivity and excellent stereochemical control. The potential of the new methodology has been investigated by synthesizing the thioether ring of a natural lantibiotic, Haloduracin ß.

Evaluation of HER2-specific peptide ligand for its employment as radiolabeled imaging probe.

HER2 transmembrane receptor is an important target in immunotherapy treatment of breast and gastroesophageal cancer. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in selection of an optimal therapy. Radiolabeled low molecular weight peptide ligands are particularly attractive as probes for molecular imaging, since they reach and bind to the target and clear from non-target organs and blood stream faster than bulky antibodies. In this study, we evaluated a potential HER2-imaging probe, an A9 nonapeptide, derived from the trastuzumab-Fab portion. Its cellular uptake was investigated by mass spectrometry analysis of the cytoplasmic cellular extracts. Moreover, based on in-silico modeling, DTPA chelator was conjugated to N-terminus of A9. 111In-labeled A9 demonstrated nanomolar affinity to HER2-expressing BT474 cells and favorable biodistribution profile in NMRI mice. This study suggests that the peptide A9 represents a good lead candidate for development of molecular probe, to be used for imaging purposes and for the delivery of cytotoxic agents.

Chemoselective Glycosylation of Peptides through S-Alkylation Reaction.

An efficient and rapid procedure for synthesizing S-linked glycopeptides is reported. The approach uses activated molecular sieves as a base to promote the selective S-alkylation of readily prepared cysteine-containing peptides, upon reaction of appropriate glycosyl halides. Considering the very mild conditions employed, the chemoselective linkage of the electrophilic sugar with a peptide sulfhydryl group occurred in satisfactory yield, allowing the incorporation of mono and disaccharide moieties. The sugar-peptide conjugates obtained from α-d-glycosyl derivatives adopt a ß-S-configuration, indicating the high stereoselectivity of the substitution reaction.

The Cysteine S-Alkylation Reaction as a Synthetic Method to Covalently Modify Peptide Sequences.

Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase.

Microwave heating in peptide side chain modification via cysteine alkylation.

Microwave irradiation has been successfully applied to a selective synthetic procedure for introducing molecular substituents on peptides, providing a noticeable reduction of the reaction time and also an increased crude peptide purity for some compounds.

Structural identification of an HER2 receptor model binding pocket to optimize lead compounds: a combined experimental and computational approach.

The structural investigation of the ligand/target interactions represents a challenging task in the field of drug discovery or lead compound optimization. In the present study, a computational approach allowed the identification of the binding site of A9 peptide, within a synthetic model of HER2 receptor (HER2-DIVMP). To this aim, molecular docking calculations and molecular dynamics simulations were employed, taking into account experimental data obtained by fluorescence studies. The computational model was further validated by performing fluorescence binding studies between the ligand A9 and HER2-DIVMP mutants, prepared by replacing key amino acid residues. A new binding pocket of HER2-DIVMP was identified, which could be fruitfully exploited for future lead-optimization studies.

Solid-Phase S-Alkylation Promoted by Molecular Sieves.

A solid-phase S-alkylation procedure to introduce chemical modification on the cysteine sulfhydryl group of a peptidyl resin is reported. The reaction is promoted by activated molecular sieves and consists of a solid-solid process, since both the catalyst and the substrate are in a solid state. The procedure was revealed to be efficient and versatile, particularly when used in combination with the solution S-alkylation approach, allowing for the introduction of different molecular diversities on the same peptide molecule.

HER2-mediated anticancer drug delivery: strategies to prepare targeting ligands highly specific for the receptor.

HER2 receptor, for its involvement in tumorigenesis, has been largely studied as topic in cancer research. In particular, the employment of trastuzumab (Herceptin), a humanized anti-HER2 antibody, showed several clinical benefits in the therapy against the breast cancer. Moreover, for its accessible extracellular domain, this receptor is considered an ideal target to deliver anticancer drugs for the receptormediated anticancer therapy. By now, monoclonal antibody and its fragments, affibody, and some peptides have been employed as targeting agents in order to deliver various drugs to HER2 positive tumor cells. In particular, the ability to perform a fast and reliable screening of a large number of peptide molecules would make possible the selection of highly specific compounds to the receptor target. In this regard, the availability of preparing a simplified synthetic model which is a good mimetic of the receptor target and can be used in a reliable screening method of ligands would be of a strategic importance for the development of selective HER2-targeting peptide molecules. Herein, we illustrate the importance of HER2-targeted anticancer therapies. We also report on a synthetic and effective mimetic of the receptor, which revealed to be a useful tool for the selection of specific HER2 ligands.

Air oxidation method employed for the disulfide bond formation of natural and synthetic peptides.

Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges.

Lipidated peptides via post-synthetic thioalkylation promoted by molecular sieves.

A thioalkylation procedure, which uses molecular sieves to promote the reaction, was exploited to provide peptides with useful functional groups (lipidic moieties), naturally occurring on proteins as post-translational modifications. The procedure was further implemented to synthesize tailor-made lipidated peptides, interesting tools to investigate biological processes involving their Ras parent proteins. Moreover, the one-pot preparation of multi-alkylated peptides confirms the versatility and flexibility of the employed methodology.

Cysteine co-oxidation process driven by native peptide folding: an example on HER2 receptor model system.

Synthetic models of receptors that have relevant biological roles are valuable tools for studying receptors itself and the corresponding ligands. Their properties can be validated at first by their capacity to fold in solution under native-like conditions and to assume conformations structurally and functionally equivalent to those in the native receptor. In this context, a new strategy to prepare the two-fragments synthetic receptor model HER2-DIVMP, an independent structural and functional motif of HER2, has been developed and the folding properties have been investigated. The strategy is based on a one-step cysteine co-oxidation procedure in slightly alkaline aqueous buffers, whereby the two separate peptide chains are allowed to self-assemble in solution. Under these conditions, the two chains spontaneously form the expected heterodimer with the correct pattern of disulfide bridges. To gain insights on the folding mechanism, we investigated the folding of two scrambled variants of the constituent peptide chains.

Chemical modifications of peptide sequences via S-alkylation reaction.

A chemoselective, convenient, and mild synthetic strategy to modify peptides on a cysteine sulfhydryl group is described. It simply requires activated molecular sieves to selectively promote S-alkylation in the presence of peptide nucleophilic functionalities. The procedure is easy to perform, fast, and provides high yields even in the case of poor electrophilic groups. Moreover, the method allows an efficient one-pot poly alkylation, proving that the sulfhydryl reactivity does not rely on its specific position within the peptide sequence.