Virtual Screening of Peptide Libraries: The Search for Peptide-Based Therapeutics Using Computational Tools.

Over the last few decades, we have witnessed growing interest from both academic and industrial laboratories in peptides as possible therapeutics. Bioactive peptides have a high potential to treat various diseases with specificity and biological safety. Compared to small molecules, peptides represent better candidates as inhibitors (or general modulators) of key protein-protein interactions. In fact, undruggable proteins containing large and smooth surfaces can be more easily targeted with the conformational plasticity of peptides. The discovery of bioactive peptides, working against disease-relevant protein targets, generally requires the high-throughput screening of large libraries, and in silico approaches are highly exploited for their low-cost incidence and efficiency. The present review reports on the potential challenges linked to the employment of peptides as therapeutics and describes computational approaches, mainly structure-based virtual screening (SBVS), to support the identification of novel peptides for therapeutic implementations. Cutting-edge SBVS strategies are reviewed along with examples of applications focused on diverse classes of bioactive peptides (i.e., anticancer, antimicrobial/antiviral peptides, peptides blocking amyloid fiber formation).

Cancer-Related Mutations in the Sam Domains of EphA2 Receptor and Ship2 Lipid Phosphatase: A Computational Study.

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.

Targeting Ship2-Sam with peptide ligands: Novel insights from a multidisciplinary approach.

The lipid phosphatase Ship2 binds the EphA2 receptor through a heterotypic Sam-Sam (Sterile alpha motif) interaction. Inhibitors of the Ship2-Sam/EphA2-Sam complex hold a certain potential as novel anticancer agents. The previously reported "KRI3" peptide binds Ship2-Sam working as a weak antagonist of the EphA2-Sam/Ship2-Sam interaction. Herein, the design and functional evaluation of KRI3 analogues, both linear and cyclic, are described. A multidisciplinary study was conducted through computational docking techniques, and conformational analyses by CD and NMR spectroscopies. The ability of new peptides to bind Ship2-Sam was analysed by NMR, MST and SPR assays. Studies on linear KRI3 analogues pointed out that aromatic interactions through tyrosines are important for the association with Ship2-Sam whereas, an increase of the net positive charge of the sequence or peptide cyclization through a disulfide bridge can favour unspecific interactions without a substantial improvement of the binding affinity to Ship2-Sam. Interestingly, preliminary cell-based assays demonstrated KRI3 cellular uptake even without the conjugation to a cell penetrating sequence with a main cytosolic localization. This work highlights important features of the KRI3 peptide that can be further exploited to design analogues able to hamper Sam-Sam interactions driven by electrostatic contacts.

Hunting for Novel Routes in Anticancer Drug Discovery: Peptides against Sam-Sam Interactions.

Among the diverse protein binding modules, Sam (Sterile alpha motif) domains attract attention due to their versatility. They are present in different organisms and play many functions in physiological and pathological processes by binding multiple partners. The EphA2 receptor contains a Sam domain at the C-terminus (EphA2-Sam) that is able to engage protein regulators of receptor stability (including the lipid phosphatase Ship2 and the adaptor Odin). Ship2 and Odin are recruited by EphA2-Sam through heterotypic Sam-Sam interactions. Ship2 decreases EphA2 endocytosis and consequent degradation, producing chiefly pro-oncogenic outcomes in a cellular milieu. Odin, through its Sam domains, contributes to receptor stability by possibly interfering with ubiquitination. As EphA2 is upregulated in many types of tumors, peptide inhibitors of Sam-Sam interactions by hindering receptor stability could function as anticancer therapeutics. This review describes EphA2-Sam and its interactome from a structural and functional perspective. The diverse design strategies that have thus far been employed to obtain peptides targeting EphA2-mediated Sam-Sam interactions are summarized as well. The generated peptides represent good initial lead compounds, but surely many efforts need to be devoted in the close future to improve interaction affinities towards Sam domains and consequently validate their anticancer properties.

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.

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.

Comparative Study of Carborane- and Phenyl-Modified Adenosine Derivatives as Ligands for the A2A and A3 Adenosine Receptors Based on a Rigid in Silico Docking and Radioligand Replacement Assay.

Adenosine receptors are involved in many physiological processes and pathological conditions and are therefore attractive therapeutic targets. To identify new types of effective ligands for these receptors, a library of adenosine derivatives bearing a boron cluster or phenyl group in the same position was designed. The ligands were screened in silico to determine their calculated affinities for the A2A and A3 adenosine receptors. An virtual screening protocol based on the PatchDock web server was developed. In the first screening phase, the effects of the functional group (organic or inorganic modulator) on the adenosine ligand affinity for the receptors were determined. Then, the lead compounds were identified for each receptor in the second virtual screening phase. Two pairs of the most promising ligands, compounds 3 and 4, and two ligands with lower affinity scores (compounds 11 and 12, one with a boron cluster and one with a phenyl group) were synthesized and tested in a radioligand replacement assay for affinity to the A2A and A3 receptors. A reasonable correlation of in silico and biological assay results was observed. In addition, the effects of a phenyl group and boron cluster, which is new adenosine modifiers, on the adenosine ligand binding were compared.

Diminished oligomerization in the synthesis of new anti-angiogenic cyclic peptide using solution instead of solid-phase cyclization.

The design and synthesis of novel peptides that inhibit angiogenesis is an important area for anti-angiogenic drug development. Cyclic and small peptides present several advantages for therapeutic application, including stability, solubility, increased bio-availability and lack of immune response in the host cell. We describe here the synthesis and biological evaluations of a new cyclic peptide analog of CBO-P11: cyclo(RIKPHE), designated herein as CBO-P23M, a hexamer peptide encompassing residues 82 to 86 of VEGF which are involved in the interaction with VEGF receptor-2. CBO-P23M was prepared using in solution cyclization, therefore reducing the peptide cyclodimerization occurred during solid-phase cyclization. The cyclic dimer of CBO-P23M, which was obtained as the main side product during synthesis of the corresponding monomer, was also isolated and investigated. Both peptides markedly reduce VEGF-A-induced phosphorylation of VEGFR-2 and Erk1/2. Moreover, they exhibit anti-angiogenic activity in an in vitro morphogenesis study. Therefore CBO-P23M and CBO-P23M dimer appear as attractive candidates for the development of novel angiogenesis inhibitors for the treatment of cancer and other angiogenesis-related diseases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 368-375, 2016.

Conformational ensembles explored dynamically from disordered peptides targeting chemokine receptor CXCR4.

This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target.

Looking for SARS-CoV-2 Therapeutics Through Computational Approaches.

BACKGROUND: In the last few years, in silico tools, including drug repurposing coupled with structure-based virtual screening, have been extensively employed to look for anti-COVID-19 agents. OBJECTIVE: The present review aims to provide readers with a portrayal of computational approaches that could be conducted more quickly and cheaply to novel anti-viral agents. Particular attention is given to docking-based virtual screening. METHODS: The World Health Organization website was consulted to gain the latest information on SARS-CoV-2, its novel variants and their interplay with COVID-19 severity and treatment options. The Protein Data Bank was explored to look for 3D coordinates of SARS-CoV-2 proteins in their free and bound states, in the wild-types and mutated forms. Recent literature related to in silico studies focused on SARS-CoV-2 proteins was searched through PubMed. RESULTS: A large amount of work has been devoted thus far to computationally targeting viral entry and searching for inhibitors of the S-protein/ACE2 receptor complex. Another large area of investigation is linked to in silico identification of molecules able to block viral proteases -including Mpro- thus avoiding maturation of proteins crucial for virus life cycle. Such computational studies have explored the inhibitory potential of the most diverse molecule databases (including plant extracts, dietary compounds, FDA approved drugs). CONCLUSION: More efforts need to be dedicated in the close future to experimentally validate the therapeutic power of in silico identified compounds in order to catch, among the wide ensemble of computational hits, novel therapeutics to prevent and/or treat COVID- 19.

EPHA2 Receptor as a Possible Therapeutic Target in Viral Infections.

BACKGROUND: The receptor tyrosine kinase EphA2 plays a role in many diseases, like cancer, cataracts, and osteoporosis. Interestingly, it has also been linked to viral infections. OBJECTIVE: Herein, current literature has been reviewed to clarify EphA2 functions in viral infections and explore its potential role as a target in antiviral drug discovery strategies. METHODS: Research and review articles and preprints connecting EphA2 to different viruses have been searched through PubMed and the web. Structures of complexes between EphA2 domains and viral proteins have been retrieved from the PDB database. RESULTS: EphA2 assumes a key role in Kaposi's sarcoma-associated herpes virus (KSHV) and Epstein Barr virus (EBV) infections by directly binding, through its ligand binding domain, viral glycoproteins. For human cytomegalovirus (HCMV), the role of EphA2 in maintaining virus latency state, through cooperation with specific viral proteins, has also been speculated. In certain cells, with high EphA2 expression levels, following ligand stimulation, receptor activation might contribute to severe symptoms accompanying a few viral infections, including lung injuries often related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). CONCLUSION: Since EphA2 works as a host receptor for certain viruses, it might be worth more deeply investigating known compounds targeting its extracellular ligand binding domain as antiviral therapeutics. Due to EphA2's function in inflammation, its possible correlation with SARS-CoV-2 cannot be excluded, but more experimental studies are needed in this case to undoubtedly attribute the role of this receptor in viral infections.

The Fight against Human Viruses: How NMR Can Help?

BACKGROUND: COVID-19 has brought the world to its knees, and there is an urgent need for new strategies to identify molecules capable of fighting the pandemic. During the last few decades, NMR (Nuclear Magnetic Resonance) spectroscopy has emerged as an intriguing structural biology instrument in the antiviral drug discovery field. OBJECTIVE: The review highlights how a variety of NMR-based tools can be employed to better understand viral machineries, develop anti-viral agents and set-up diagnostic and therapeutic routes. METHODS: Works summarized herein were searched through PubMed database and the Web. RESULTS: The review focuses on a subset of human viruses that have been largely studied through NMR techniques. Indeed, NMR solid- or solution-state methodologies allow to gain structural information on viral proteins and viral genomes either in isolation or bound to diverse binding partners. NMR data can be employed to set up structure-based approaches to design efficient antiviral agents inhibiting crucial steps of viral life cycle. In addition, NMR-based metabolomics analyses of biofluids from virus-infected patients let identify metabolites biomarkers of the disease and follow changes in metabolic profiles associated with antiviral therapy thus paving the way for novel diagnostic and therapeutic approaches. CONCLUSION: Considering the NMR-based work conducted on different viruses, we believe that in the near future, much more NMR efforts will be devoted to discovering novel anti SARS-CoV-2 agents.

Protein Interaction Domains: Structural Features and Drug Discovery Applications (Part 2).

BACKGROUND: Proteins present a modular organization made up of several domains. Apart from the domains playing catalytic functions, many others are crucial to recruit interactors. The latter domains can be defined as "PIDs" (Protein Interaction Domains) and are responsible for pivotal outcomes in signal transduction and a certain array of normal physiological and disease-related pathways. Targeting such PIDs with small molecules and peptides able to modulate their interaction networks, may represent a valuable route to discover novel therapeutics. OBJECTIVE: This work represents a continuation of a very recent review describing PIDs able to recognize post-translationally modified peptide segments. On the contrary, the second part concerns with PIDs that interact with simple peptide sequences provided with standard amino acids. METHODS: Crucial structural information on different domain subfamilies and their interactomes was gained by a wide search in different online available databases (including the PDB (Protein Data Bank), the Pfam (Protein family), and the SMART (Simple Modular Architecture Research Tool)). Pubmed was also searched to explore the most recent literature related to the topic. RESULTS AND CONCLUSION: PIDs are multifaceted: they have all diverse structural features and can recognize several consensus sequences. PIDs can be linked to different diseases onset and progression, like cancer or viral infections and find applications in the personalized medicine field. Many efforts have been centered on peptide/peptidomimetic inhibitors of PIDs mediated interactions but much more work needs to be conducted to improve drug-likeness and interaction affinities of identified compounds.

NMR Spectroscopy in the Conformational Analysis of Peptides: An Overview.

BACKGROUND: NMR spectroscopy is one of the most powerful tools to study the structure and interaction properties of peptides and proteins from a dynamic perspective. Knowing the bioactive conformations of peptides is crucial in the drug discovery field to design more efficient analogue ligands and inhibitors of protein-protein interactions targeting therapeutically relevant systems. OBJECTIVE: This review provides a toolkit to investigate peptide conformational properties by NMR. METHODS: Articles cited herein, related to NMR studies of peptides and proteins were mainly searched through PubMed and the web. More recent and old books on NMR spectroscopy written by eminent scientists in the field were consulted as well. RESULTS: The review is mainly focused on NMR tools to gain the 3D structure of small unlabeled peptides. It is more application-oriented as it is beyond its goal to deliver a profound theoretical background. However, the basic principles of 2D homonuclear and heteronuclear experiments are briefly described. Protocols to obtain isotopically labeled peptides and principal triple resonance experiments needed to study them, are discussed as well. CONCLUSION: NMR is a leading technique in the study of conformational preferences of small flexible peptides whose structure can be often only described by an ensemble of conformations. Although NMR studies of peptides can be easily and fast performed by canonical protocols established a few decades ago, more recently we have assisted to tremendous improvements of NMR spectroscopy to investigate instead large systems and overcome its molecular weight limit.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications.

BACKGROUND: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as Protein Interaction Domains (PIDs). OBJECTIVE: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. METHOD: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns were retrieved through Pubmed and analyzed. RESULTS AND CONCLUSION: PIDs are rather versatile as concerned with their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

Sam Domains in Multiple Diseases.

BACKGROUND: The sterile alpha motif (Sam) domain is a small helical protein module, able to undergo homo- and hetero-oligomerization, as well as polymerization, thus forming different types of protein architectures. A few Sam domains are involved in pathological processes and consequently, they represent valuable targets for the development of new potential therapeutic routes. This study intends to collect state-of-the-art knowledge on the different modes by which Sam domains can favor disease onset and progression. METHODS: This review was build up by searching throughout the literature, for: a) the structural properties of Sam domains, b) interactions mediated by a Sam module, c) presence of a Sam domain in proteins relevant for a specific disease. RESULTS: Sam domains appear crucial in many diseases including cancer, renal disorders, cataracts. Often pathologies are linked to mutations directly positioned in the Sam domains that alter their stability and/or affect interactions that are crucial for proper protein functions. In only a few diseases, the Sam motif plays a kind of "side role" and cooperates to the pathological event by enhancing the action of a different protein domain. CONCLUSION: Considering the many roles of the Sam domain into a significant variety of diseases, more efforts and novel drug discovery campaigns need to be engaged to find out small molecules and/or peptides targeting Sam domains. Such compounds may represent the pillars on which to build novel therapeutic strategies to cure different pathologies.

About TFE: Old and New Findings.

The fluorinated alcohol 2,2,2-Trifluoroethanol (TFE) has been implemented for many decades now in conformational studies of proteins and peptides. In peptides, which are often disordered in aqueous solutions, TFE acts as secondary structure stabilizer and primarily induces an α -helical conformation. The exact mechanism through which TFE plays its stabilizing roles is still debated and direct and indirect routes, relying either on straight interaction between TFE and molecules or indirect pathways based on perturbation of solvation sphere, have been proposed. Another still unanswered question is the capacity of TFE to favor in peptides a bioactive or a native-like conformation rather than simply stimulate the raise of secondary structure elements that reflect only the inherent propensity of a specific amino-acid sequence. In protein studies, TFE destroys unique protein tertiary structure and often leads to the formation of non-native secondary structure elements, but, interestingly, gives some hints about early folding intermediates. In this review, we will summarize proposed mechanisms of TFE actions. We will also describe several examples, in which TFE has been successfully used to reveal structural properties of different molecular systems, including antimicrobial and aggregation-prone peptides, as well as globular folded and intrinsically disordered proteins.

Synthesis and Antiproliferative Effect of Ethyl 4-[4-(4-Substituted Piperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxalinecarboxylate Derivatives on Human Leukemia Cells.

Acute leukemia is a hematological malignancy with high incidence and recurrence rates and is characterized by an accumulation of blasts in bone marrow due to proliferation of immature lymphoid or myeloid cells associated with a blockade of differentiation. The heterogeneity of leukemia led us to look for new specific molecules for leukemia subtypes or for therapy-resistant cases. Among heterocyclic derivatives that attracted attention due to their wide range of biological activities, we focused our interest on the pyrrolo[1,2-a]quinoxaline heterocyclic framework that has been previously identified as an interesting scaffold for antiproliferative activities against various human cancer cell lines. In this work, new ethyl 4-[4-(4-substituted piperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxalinecarboxylate derivatives (1 a-o) were designed, synthesized, and evaluated against five different leukemia cell lines, including Jurkat and U266 (lymphoid cell lines) and K562, U937, and HL60 (myeloid cell lines), as well as on normal human peripheral blood mononuclear cells (PBMCs). This new pyrrolo[1,2-a]quinoxaline series showed interesting cytotoxic potential against all tested leukemia cell lines. In particular, pyrroloquinoxalines 1 a and 1 m,n seem to be interesting due to their high activity against leukemia and their low activity against normal hematopoietic cells, leading to a high index of selectivity.

Synthesis and evaluation of the cytotoxic activity of novel ethyl 4-[4-(4-substitutedpiperidin-1-yl)]benzyl-phenylpyrrolo[1,2-a]quinoxaline-carboxylate derivatives in myeloid and lymphoid leukemia cell lines.

Leukemia is the most common blood cancer, and its development starts at diverse points, leading to distinct subtypes that respond differently to therapy. This heterogeneity is rarely taken into account in therapies, so it is still essential to look for new specific drugs for leukemia subtypes or even for therapy-resistant cases. Among heterocyclic compounds that attracted a lot of attention because of its wide spread biological activities, the pyrrolo[1,2-a]quinoxaline heterocyclic framework has been identified as interesting scaffolds for antiproliferative activity against various human cancer cell lines. In the present study, novel ethyl 4-[4-(4-substitutedpiperidin-1-yl)]benzyl-phenylpyrrolo[1,2-a]quinoxaline-carboxylate derivatives 1a-l have been designed and synthesized. Their cytotoxicities were evaluated against five different leukemia cell lines, including Jurkat and U266 (lymphoid cell lines), and K562, U937, HL60 (myeloid cell lines), as well as normal human peripheral blood mononuclear cells (PBMNCs). Then, apoptosis study was performed with the more interesting compounds. The new pyrrolo[1,2-a]quinoxaline series showed promising cytotoxic potential against all leukemia cell lines tested, and some compounds showed better results than the reference compound A6730. Some compounds, such as 1a, 1e, 1g and 1h are promising because of their high activity against leukemia and their low activity against normal hematopoietic cells. Structure-activity relationships of these new synthetic compounds 1a-l are here also discussed.

Intrinsically disordered amphiphilic peptides as potential targets in drug delivery vehicles.

Intrinsically disordered proteins/peptides play a crucial role in many physiological and pathological events and may assume a precise conformation upon binding to a specific target. Recently, we have described the conformational and functional properties of two linear ester peptides provided with the following sequences: Y-G-E-C-P-C-K-OAllyl (PepK) and Y-G-E-C-P-C-E-OAllyl (PepE). Both peptides are characterized by the presence of the "CPC" motif together with a few amino acids able to promote disorder. The CPC sequence is a binding motif for the CXCR4 receptor that represents a well-known target for cancer therapies. In this paper, we report on synthetic amphiphilic peptides that consist of lipophilic derivatives of PepE and PepK bearing two stearic alkyl chains and/or an ethoxylic spacer. These peptide amphiphiles form stable supramolecular aggregates; they present conformational features that are typical of intrinsically disordered molecules as shown by CD spectroscopy. Solution fluorescence and DLS studies have been performed to evaluate Critical Micellar Concentrations and the dimension of supramolecular aggregates. Moreover, preliminary in vitro cell-based assays have been conducted to investigate the molecular recognition processes involving the CXCR4 receptor. In the end, the results obtained have been compared with the previous data generated by the corresponding non-amphiphilic peptides (PepE and PepK).