Antimicrobial peptides (AMPs) show great therapeutic potential due to their unique mechanism of action that guarantees broad-spectrum efficacy and limits bacterial antibiotic resistance. However, challenges such as limited stability and cytotoxicity toward host cells still limit their clinical translation, highlighting the need for new approaches, such as size reduction and lipid conjugation, to enhance their efficacy, cell penetration, stability, and safety. Herein, we report the de novo design of a library of ultra-short lipopeptides based on a rigid l-Arg-l-Pro-l-Arg core, conceived to control conformational restriction and amphiphilic organization rather than mimicking longer natural AMPs. The compounds were synthesized and preliminarily evaluated in vitro against 3 Gram-negative and 3 Gram-positive strains. Systematic modulation of lipid positioning and linker orientation in this minimal scaffold led to the identification of promising candidates displaying MIC values in the low-μM range against both gram-negative and gram-positive bacteria. Of the newly developed compounds, 15 exhibited optimal lipophilicity, excellent human-serum stability and a favourable safety profile, showing only low to moderate toxicity toward renal, hepatic, and red blood cells. Additionally, 15 proved effective in reducing S. aureus biofilm formation and showed strong activity against five clinical isolates. It acts as a bacteriostatic agent by perturbing bacterial membrane integrity, positioning it as a promising starting point for the development of a new class of chemotypes that could offer an alternative strategy for treating infections caused by this gram-positive pathogen.

The selective manipulation of cysteine residues in solid-phase peptide synthesis (SPPS), beyond disulfide bridge formation, remains challenging, and an efficient approach for the removal of the widely used trityl (Trt) protecting group under solid-phase conditions is still lacking. To address this issue, herein, a thiophilic transition metal-mediated strategy for the selective S -detritylation of cysteine on resin was developed by modifying a threecomponent catalytic system originally conceived for solution synthesis. The systematic screening of copper, zinc, and silver salts led to the identification of Ag(CF 3 CO 2 ) and AgNO 3 as the most effective promoters of Trt removal. Reaction conditions were optimized to afford reproducible and efficient deprotection, and mechanistic insights were obtained through control experiments. The protocol proved compatible with common protecting groups and environmentally benign solvent systems. Finally, in situ thiol release enabled direct on-resin S -alkylation with a range of aliphatic substituents, including those bearing aromatic moieties, demonstrating the synthetic utility of the method at a new level of orthogonality for late-stage functionalization of cysteine containing resin-bound peptides.

Non-small cell lung cancer (NSCLC), the leading cause of cancer-related mortality worldwide, poses a formidable challenge due to its heterogeneity and the emergence of resistance to targeted therapies. While initially effective, first- and third-generation EGFR-tyrosine kinase inhibitors (TKIs) often fail to control disease progression, leaving patients with limited treatment options. To address this unmet medical need, we explored the therapeutic potential of multitargeting agents that simultaneously inhibit two key signalling pathways, the mesenchymal-epithelial transition factor (c-MET) and the G protein-coupled receptor Smoothened (SMO), frequently dysregulated in NSCLC. By employing a combination of in silico drug repurposing and structure-based structure-activity relationship (SAR) studies, we identified and developed novel c-MET/SMO-targeting agents with antiproliferative activity against first- as well as third-generation EGFR-TKI-resistant NSCLC cells suggesting a synergistic effect arising from the simultaneous inhibition of c-MET and SMO.

Vaccinology has revolutionized modern medicine, delivering groundbreaking solutions to prevent and control infectious diseases while pioneering innovative strategies to tackle non-infectious challenges, including cancer. Traditional vaccines faced inherent limitations, driving the evolution of next-generation vaccines such as subunit vaccines, peptide-based vaccines, and nucleic acid-based platforms. Among these, nucleic acid-based vaccines, including DNA and mRNA technologies, represent a major innovation. Pioneering studies in the 1990s demonstrated their ability to elicit immune responses by encoding specific antigens. Recent advancements in delivery systems and molecular engineering have overcome initial challenges, enabling their rapid development and clinical success. This review explores nucleic acid-based vaccines, including chemically modified variants, by examining their mechanisms, structural features, and therapeutic potential, while underscoring their pivotal role in modern immunization strategies and expanding applications across contemporary medicine.

The integration of low-frequency ultrasound with Solid-Phase Peptide Synthesis (SPPS) was explored to establish a Sustainable Ultrasound-assisted Solid-Phase Peptide Synthesis (SUS-SPPS) method. This innovative approach significantly reduces solvent consumption, washing steps, time, and reagent usage compared to conventional manual SPPS protocols. The SUS-SPPS method exploits ultrasound at every stage of synthesis and work-up, reducing the process to just two steps. The first step sequentially combines Fmoc-amino acid coupling, capping of unreacted amino groups, and Fmoc deprotection into a single operation, while the second one consists of a single washing procedure. Moreover, we demonstrated that the method is compatible with various resin types, including Rink-amide, Wang, and Cl-Trt resins, and facilitates the efficient synthesis of peptides of varying lengths (up to 20-mers) and compositions, including those traditionally considered "difficult sequences", with excellent yields and purity. Notably, SUS-SPPS reduces solvent usage per coupling cycle by 83-88%, marking a significant breakthrough in sustainable peptide synthesis.

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.

The present study describes a small library of peptides derived from a potent and selective CXCR4 antagonist (3), wherein the native disulfide bond is replaced using a side-chain to tail macrolactamization technique to vary ring size and amino acid composition. The peptides were preliminary assessed for their ability to interfere with the interaction between the receptor and anti-CXCR4 PE-conjugated antibody clone 12G5. Two promising candidates (13 and 17) were identified and further evaluated in a 125I-CXCL12 competition binding assay, exhibiting IC50 in the low-nanomolar range. Furthermore, both candidates displayed high selectivity towards CXCR4 with respect to the cognate receptor CXCR7, ability to block CXCL12-dependent cancer cell migration, and receptor internalization, albeit at a higher concentration compared to 3. Molecular modeling studies on 13 and 17 produced a theoretical model that may serve as a guide for future modifications, aiding in the development of analogs with improved affinity. Finally, the study provides valuable insights into developing therapeutic agents targeting CXCR4-mediated processes, demonstrating the adaptability of our lead peptide 3 to alternative cyclization approaches and offering prospects for comprehensive investigations into the receptor region's interaction with its C-terminal region.

Macrocyclization presents a valuable strategy for enhancing the pharmacokinetic and pharmacodynamic profiles of short bioactive peptides. The exploration of various macrocyclic characteristics, such as crosslinking tethers, ring size, and orientation, is generally conducted during the early stages of development. Herein, starting from a potent and selective C-X-C chemokine receptor 4 (CXCR4) cyclic heptapeptide antagonist mimicking the N-terminal region of CXCL12, we demonstrated that the disulfide bridge could be successfully replaced with a side-chain to side-chain lactam bond, which is commonly not enlisted among the conventional disulfide mimetics. An extensive investigation was carried out to explore the chemical space of the resulting peptides, including macrocyclization width, stereochemical configuration, and lactam orientation, all of which were correlated with biochemical activity. We identified a novel heptapeptide that fully replicates the pharmacological profile of the parent peptide on CXCR4, including its potency, selectivity, and antagonistic activity, while demonstrating enhanced stability in a reductive environment. At this stage, computational studies were instructed to shed light on how the lactam cyclization features influenced the overall structure of 21 and, in turn, its ability to interact with the receptor. We envisage that these findings can give new momentum to the use of lactam cyclization as a disulfide bond mimetic and contribute to the enhancement of the repertoire for peptide-based drug development, thereby paving the way for novel avenues in therapeutic innovation.

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.

Abstract In the field of modern biomedicine, significant progress has been made in countering or restoring faulty genes using exogenous nucleic acids or their mimetics (NAMs). This approach enables targeted and long-lasting therapeutic effects by circumventing the need of interventions at a protein level. Despite the boom of clinical approvals, the development of NAMs still faces crucial challenges, including susceptibility to metabolic breakdown, limited uptake by cells, self-aggregation, and the potential for immune reactions. In addition to employing advanced delivery strategies, alternative methods to address these issues involve modifying NAMs chemically or combining oligonucleotides (ONs) with cellular carriers, including cell-penetrating peptides. Against this background, herein we present the nucleopeptides (NPs) as a family of unexplored NAMs that bridge the gap between ONs and peptide structures. By incorporating two consecutive amino acids—one bearing a nucleobase on the side chain and the other remaining unaltered—we created a monomer that spans the length of a single nucleotide. This functional module possesses nucleobase recognition elements along with additional functional groups eligible to modulate physio-chemical properties. We investigated this class of macromolecules by preliminarily devising an efficient solid-phase peptide synthesis technique profitable for homo- and hetero-sequences and sparing the monomer assembly in solution. [1] The influence of the amino acid composition on target binding properties, metabolic stability, and cellular penetration, were assessed through the synthesis of a series of homothymine hexameric derivatives. This simplified model was also used to investigate the distance between the nucleobase and the backbone, the absolute configuration of the nucleobase- bearing element, and the polycationic/anionic nature of the unmodified amino acid. [2-4] We evaluated the recognition and the thermodynamic stability of NPs/DNA and NPs/RNA hetero-duplexes through circular dichroism and UV spectroscopy experiments, while we employed confocal microscopy with suitable FITC- labeled derivatives to determine the intracellular localization in target cells. [1,3] Our results suggested that NPs exhibit distinct binding properties towards complementary RNA and DNA sequences according to their aminoacidic composition, and are characterized by a higher affinity compared to ONs and PNAs, thus arising as promising cell penetrating NAMs. [1] Mercurio ME, Tomassi S, et al. J Org Chem. 2016, 81(23), 11612- 11625. [2] Tomassi S, Ieranò C, et al. Bioorg Med Chem. 2018, 26(9), 2539-2550. [3] Tomassi S, Montalban FF, et al. Symmetry 2019, 11(4), 567-582. [4] Tomassi S, Ieranò C, et al. Int J Mol Sci. 2022, 23(15), 8504-8518. Citation Format: Stefano Tomassi, Caterina Ieranò, Stefania Scala, Anna Messere, Salvatore Di Maro. Synthesis and structure-activity studies of DNA- and RNA-binding nucleopeptides with a cell penetrating potential [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A026.

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.

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.

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.

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.

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.

Nucleopeptides represent an intriguing class of nucleic acid analogues, in which nucleobases are placed in a peptide structure. The incorporation of D- and/or L-amino acids in nucleopeptide molecules allows the investigation of the role of backbone stereochemistry in determining the formation of DNA and RNA hybrids. Circular Dichroism (CD) spectroscopic studies indicated the nucleopeptide as having fully l-backbone configuration-formed stable hybrid complexes with RNA molecules. Molecular Dynamics (MD) simulations suggested a potential structure of the complex resulting from the interaction between the l-nucleopeptide and RNA strand. From this study, both the backbone (ionics and H-bonds) and nucleobases (pairing and π-stacking) of the chiral nucleopeptide appeared to be involved in the hybrid complex formation, highlighting the key role of the backbone stereochemistry in the formation of the nucleopeptide/RNA complexes.

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.

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.