The bright side of chemistry: Exploring synthetic peptide-based anticancer vaccines.

The present review focuses on synthetic peptide-based vaccine strategies in the context of anticancer intervention, paying attention to critical aspects such as peptide epitope selection, adjuvant integration, and nuanced classification of synthetic peptide cancer vaccines. Within this discussion, we delve into the diverse array of synthetic peptide-based anticancer vaccines, each derived from tumor-associated antigens (TAAs), including melanoma antigen recognized by T cells 1 (Melan-A or MART-1), mucin 1 (MUC1), human epidermal growth factor receptor 2 (HER-2), tumor protein 53 (p53), human telomerase reverse transcriptase (hTERT), survivin, folate receptor (FR), cancer-testis antigen 1 (NY-ESO-1), and prostate-specific antigen (PSA). We also describe the synthetic peptide-based vaccines developed for cancers triggered by oncovirus, such as human papillomavirus (HPV), and hepatitis C virus (HCV). Additionally, the potential synergy of peptide-based vaccines with common therapeutics in cancer was considered. The last part of our discussion deals with the realm of the peptide-based vaccines delivery, highlighting its role in translating the most promising candidates into effective clinical strategies. Although this discussion does not cover all the ongoing peptide vaccine investigations, it aims at offering valuable insights into the chemical modifications and the structural complexities of anticancer peptide-based vaccines.

Ultrasound-assisted Peptide Nucleic Acids synthesis (US-PNAS).

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.

Tailoring the Structure of Cell Penetrating DNA and RNA Binding Nucleopeptides.

Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these molecules into drugs is a long and difficult process that justifies the continuous research of new chemotypes endowed with favorable binding, pharmacokinetic and pharmacodynamic properties. In this scenario, we describe the synthesis of two sets of homo-thymine nucleopeptides, in which nucleobases are inserted in a peptide structure, to investigate the role of the underivatized amino acid residue and the distance of the nucleobase from the peptide backbone on the nucleic acid recognition process. It is worth noting that the CD spectroscopy investigation showed that two of the reported nucleopeptides, consisting of alternation of thymine functionalized L-Orn and L-Dab and L-Arg as underivatized amino acids, were able to efficiently bind DNA and RNA targets and cross both cell and nuclear membranes.

CLIPSing Melanotan-II to Discover Multiple Functionally Selective hMCR Agonists.

The pleiotropic role played by melanocortin receptors (MCRs) in both physiological and pathological processes has stimulated medicinal chemists to develop synthetic agonists/antagonists with improved potency and selectivity. Here, by deploying the Chemical Linkage of Peptide onto Scaffolds strategy, we replaced the lactam cyclization of melanotan II (MT-II), a potent and unselective agonist of human MCRs (hMCRs), with different xylene-derived thioethers. The newly designed peptides displayed binding affinities toward MCRs ranging from the low nanomolar to the sub-micromolar range, highlighting a correlation between the explored linkers and the affinity toward hMCRs. In contrast to the parent peptide (MT-II), compound 5 displayed a remarkable functional selectivity toward the hMC1R. Enhanced sampling molecular dynamics simulations were found to be instrumental in outlining how the employed cyclization strategy affects the peptides' conformational behavior and, as a consequence, the detected hMC1R affinity. Additionally, a model of the peptide 5/hMC1R complex employing the very recently reported cryogenic electron microscopy receptor structure was provided.

Lead Discovery of SARS-CoV-2 Main Protease Inhibitors through Covalent Docking-Based Virtual Screening.

During almost all 2020, coronavirus disease 2019 (COVID-19) pandemic has constituted the major risk for the worldwide health and economy, propelling unprecedented efforts to discover drugs for its prevention and cure. At the end of the year, these efforts have culminated with the approval of vaccines by the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA) giving new hope for the future. On the other hand, clinical data underscore the urgent need for effective drugs to treat COVID-19 patients. In this work, we embarked on a virtual screening campaign against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro chymotrypsin-like cysteine protease employing our in-house database of peptide and non-peptide ligands characterized by different types of warheads acting as Michael acceptors. To this end, we employed the AutoDock4 docking software customized to predict the formation of a covalent adduct with the target protein. In vitro verification of the inhibition properties of the most promising candidates allowed us to identify two new lead inhibitors that will deserve further optimization. From the computational point of view, this work demonstrates the predictive power of AutoDock4 and suggests its application for the in silico screening of large chemical libraries of potential covalent binders against the SARS-CoV-2 Mpro enzyme.

Disulfide Bond Replacement with 1,4- and 1,5-Disubstituted [1,2,3]-Triazole on C-X-C Chemokine Receptor Type 4 (CXCR4) Peptide Ligands: Small Changes that Make Big Differences.

Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.

Boosting Fmoc Solid-Phase Peptide Synthesis by Ultrasonication.

We investigated the ultrasonication-mediated effects on the Fmoc-based solid-phase peptide synthesis (SPPS). Our study culminated with the development of an ultrasound-assisted strategy (US-SPPS) that allowed for the synthesis of different biologically active peptides (up to 44-mer), with a remarkable savings of material and reaction time. Noteworthy, ultrasonic irradiation did not exacerbate the main side reactions and improved the synthesis of peptides endowed with "difficult sequences", placing the US-SPPS among the current high-efficient peptide synthetic strategies.

Cationic nucleopeptides as novel non-covalent carriers for the delivery of peptide nucleic acid (PNA) and RNA oligomers.

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.

Structure-Activity Relationships and Biological Characterization of a Novel, Potent, and Serum Stable C-X-C Chemokine Receptor Type 4 (CXCR4) Antagonist.

In our ongoing pursuit of CXCR4 antagonists as potential anticancer agents, we recently developed a potent, selective, and plasma stable peptide, Ac-Arg-Ala-[d-Cys-Arg-Phe-Phe-Cys]-COOH (3). Nevertheless, this compound was still not potent enough (IC50 ≈ 53 nM) to enter preclinical studies. Thus, a lead-optimization campaign was here undertaken to further improve the binding affinity of 3 while preserving its selectivity and proteolytic stability. Specifically, extensive structure-activity relationships (SARs) investigations were carried out on both its aromatic and disulfide forming amino acids. One among the synthesized analogue, Ac-Arg-Ala-[d-Cys-Arg-Phe-His-Pen]-COOH (19), displayed subnanomolar affinity toward CXCR4, with a marked selectivity over CXCR3 and CXCR7. NMR and molecular modeling studies disclosed the molecular bases for the binding of 19 to CXCR4 and for its improved potency compared to the lead 3. Finally, biological assays on specific cancer cell lines showed that 19 can impair CXCL12-mediated cell migration and CXCR4 internalization more efficiently than the clinically approved CXCR4 antagonist plerixafor.

Computer-Aided Identification and Lead Optimization of Dual Murine Double Minute 2 and 4 Binders: Structure-Activity Relationship Studies and Pharmacological Activity.

The function of p53 protein, also known as "genome guardian", might be impaired by the overexpression of its primary cellular inhibitor, the murine double minute 2 protein (MDM2). However, the recent finding that MDM2-selective inhibitors induce high levels of its homologue MDM4, prompt us to identify, through a receptor-based virtual screening on an in house database, dual MDM2/MDM4 binders. Compound 1 turned out to possess an IC50 of 93.7 and of 4.6 nM on MDM2 and MDM4, respectively. A series of compounds were synthesized to optimize its activity on MDM2. As a result, compound 12 showed low nanomolar IC50 for both targets. NMR studies confirmed the pocket of binding of 12 as predicted by the Glide docking software. Notably, 12 was able to cause concentration-dependent inhibition of cell proliferation, yielding an IC50 value of 356 ± 21 nM in neuroblastoma SHSY5Y cells and proved even to efficiently block cancer stem cell growth.

Exploiting the 4-Phenylquinazoline Scaffold for the Development of High Affinity Fluorescent Probes for the Translocator Protein (TSPO).

The quinazoline class was exploited to search for a new translocator protein (TSPO) fluorescent probe endowed with improved affinity and residence time (RT). Computational studies on an "in-house" collection of quinazoline derivatives, featuring highly steric demanding groups at the amide nitrogen, suggested that, despite their molecular extension, these ligands are still easily lodged in the TSPO binding site. Binding assays supported this hypothesis, highlighting a low nanomolar/subnanomolar affinity of these ligands, together with a higher RT of the representative compound 11 with respect to our previously reported indole-based fluorescent probe. Thanks to the amenability of the amide nitrogen atom to be substituted with bulky groups, we developed quinazoline-based imaging tools by fluorescently labeling the scaffold at this position. Probes with relevant TSPO affinity, favorable spectroscopic properties, and improved RT were identified. The results from fluorescence microscopy showed that these probes specifically labeled the TSPO at the mitochondrial level in the U343 cell line.

Dual MET and SMO Negative Modulators Overcome Resistance to EGFR Inhibitors in Human Nonsmall Cell Lung Cancer.

Tyrosine kinase inhibitors (TKIs) of the EGF receptor (EGFR) have provided a significant improvement in the disease outcome of nonsmall cell lung cancer (NSCLC). Unfortunately, resistance to these agents frequently occurs, and it is often related to the activation of the Hedgehog (Hh) and MET signaling cascades driving the epithelial-to-mesenchymal transition (EMT). Because the concomitant inhibition of both Hh and MET pathways restores the sensitivity to anti-EGFR drugs, here we aimed at discovering the first compounds that block simultaneously MET and SMO. By using an "in silico drug repurposing" approach and by validating our predictions both in vitro and in vivo, we identified a set of compounds with the desired dual inhibitory activity and enhanced antiproliferative activity on EGFR TKI-resistant NSCLC. The identification of the known MET TKIs, glesatinib and foretinib, as negative modulators of the Hh pathway, widens their application in the context of NSCLC.

Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides.

Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane.

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis.

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies.

Chemical modifications in the seed region of miRNAs 221/222 increase the silencing performances in gastrointestinal stromal tumor cells.

Most GastroIntestinal Stromal Tumors (GISTs) are characterized by KIT gene overexpression, which in turn is regulated by levels of microRNA 221 and microRNA 222. GISTs can also be distinguished by their miRNAs expression profile in which miRNAs 221/222 result reduced in comparison with GI normal tissues. In this paper, to restore normal miRNAs levels and to improve the silencing performances of miRNAs 221/222, new miRNA mimics in which guide strands are modified by Phosphorothioate (PS) and/or 2'-O-methyl RNA (2'-OMe) inside and outside the seed region, were synthesized and tested in GIST48 cells. We evaluated the positional effect of the chemical modifications on the miRNAs silencing activity, compared to natural and several commercial miRNA mimics. Our results show that chemically modified miRNAs 221/222 with alternating 2'-OMe-PS and natural nucleotides in the seed region are effective inhibitors of KIT gene expression and exhibit increased stability in rat plasma. Besides, their transfection in GIST 48 cells showed significant effects on different cellular processes in which KIT plays a functional role for tumor development (such as migration, cell proliferation, and apoptosis). Therefore, modified miRNAs 221/222 may provide an alternative therapeutic option for GIST treatment also aimed to overcome drug resistance concerns.

Hairpin oligonucleotides forming G-quadruplexes: new aptamers with anti-HIV activity.

We describe the facile syntheses of new modified oligonucleotides based on d(TG3AG) that form bimolecular G-quadruplexes and possess a HEG loop as an inversion of polarity site 3'-3' or 5'-5' and aromatic residues conjugated to the 5'-end through phosphodiester bonds. The conjugated hairpin G-quadruplexes exhibited parallel orientation, high thermal stability, elevated resistance in human serum and high or moderate anti-HIV-1 activity with low cytotoxicity. Further, these molecules showed significant binding to HIV envelope glycoproteins gp120, gp41 and HSA, as revealed by SPR assays. As a result, these conjugated hairpins represent the first active anti-HIV-1 bimolecular G-quadruplexes based on the d(TG3AG) sequence.

Synthesis and gene silencing properties of siRNAs containing terminal amide linkages.

The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3' end, carrying 5'-phosphate and 3'-hydroxyl groups (siRNAs). Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3' overhang recognition by the PAZ domain and the 5'-phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA) moieties at 5', and at 3' positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs.

Synthesis, biophysical characterization and anti-HIV activity of d(TG3AG) Quadruplexes bearing hydrophobic tails at the 5'-end.

Novel conjugated G-quadruplex-forming d(TG3AG) oligonucleotides, linked to hydrophobic groups through phosphodiester bonds at 5'-end, have been synthesized as potential anti-HIV aptamers, via a fully automated, online phosphoramidite-based solid-phase strategy. Conjugated quadruplexes showed pronounced anti-HIV activity with some preference for HIV-1, with inhibitory activity invariably in the low micromolar range. The CD and DSC monitored thermal denaturation studies on the resulting quadruplexes, indicated the insertion of lipophilic residue at the 5'-end, conferring always improved stability to the quadruplex complex (20<ΔTm<40°C). The data suggest no direct functional relationship between the thermal stability and anti-HIV activity of the folded conjugated G-quartets. It would appear that the nature of the residue at 5' end of the d(TG3AG) quadruplexes plays an important role in the thermodynamic stabilization but a minor influence on the anti-HIV activity. Moreover, a detailed CD and DSC analyses indicate a monophasic behaviour for sequences I and V, while for ODNs (II-IV) clearly show that these quadruplex structures deviate from simple two-state melting, supporting the hypothesis that intermediate states along the dissociation pathway may exist.

Synthesis and biological properties of caffeic acid-PNA dimers containing guanine.

Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is endowed with high antioxidant activity. CA derivatives (such as amides) have gained a lot of attention due to their antioxidative, antitumor and antimicrobial properties as well as stable characteristics. Caffeoyl-peptide derivatives showed different antioxidant activity depending on the type and the sequence of amino acid used. For these reasons, we decided to combine CA with Peptide Nucleic Acid (PNA) to test whether the new PNA-CA amide derivatives would result in an improvement or gain of CA's biological (i.e., antioxidant, cytotoxic, cytoprotective) properties. We performed the synthesis and characterization of seven dimer conjugates with various combinations of nucleic acid bases and focused NMR studies on the model compound ga-CA dimer. We demonstrate that PNA dimers containing guanine conjugated to CA exhibited different biological activities depending on composition and sequence of the nucleobases. The dimer ag-CA protected HepG2, SK-B-NE(2), and C6 cells from a cytotoxic dose of hydrogen peroxide (H2O2).

Tuning RNA Interference by Enhancing siRNA/PAZ Recognition.

Chemically modified siRNAs were synthesized to enhance the corresponding silencing activities. The introduced modifications endowed siRNAs with high silencing effect, long RNAi persistence, and better serum resistance. Theoretical data allowed us to correlate the observed siRNAs interfering performance with the peculiar interactions with PAZ.