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.

From the approximately 800 members of the G protein-coupled receptor (GPCR) family, more than 100 remain orphans (oGPCRs). There is evidence indicating that some oGPCRs may carry out a physiological role independently from endogenous ligands; this includes forming heteromers with other GPCRs and altering their functional and pharmacological properties via allosteric interactions. Recent studies have shown that some of these oGPCRs, e.g. GPR88 and GPR139, allosterically inhibit opioid activity by interacting with the μ-opioid receptor (μOR). Here, we have focused on the characterization of the interaction between GPR88 and µOR and the allosteric mechanism of inhibition. We confirmed that GPR88 inhibits µOR function in striatal neuronal primary cultures. Moreover, using a peptide-interfering approach combined with biophysical and biochemical techniques, we identified that GPR88 and µOR interact via transmembrane helix 6. A combination of molecular dynamic simulations and site-directed mutagenesis have allowed to propose that the negative regulatory role of GPR88 on µOR is due to the Q2986.49 side chain of GPR88.

In this paper we report a novel acid-modulated strategy for peptide microarray production on biosensor interfaces. We have initially selected controlled pore glass (CPG) as support for solid phase peptide synthesis (SPPS) to implement a chemistry that can be efficiently performed at the interface of multiple FET sensors, eventually to generate label-free peptide microarrays for protein screening. Our chemistry uses temporary protection of the N-terminal amino function of each amino acid building block with a tert-butyloxycarbonyl (Boc) group that can be removed after each SPPS cycle, in combination with semi-permanent protection of the side chains of trifunctional amino acid residues. Such protection scheme, with a well-proven record of application in conventional, batchwise SPPS, has been fine tuned for optimal performance on CPG and, from there, translated to SPR chips that allow layer-by-layer monitoring of amino acid coupling. Our results validate this acid-modulated synthesis as a feasible approach for producing peptides in high yield and purity on flat glass surfaces such as those in bio-FETs.

Glycosaminoglycan (GAG)-binding proteins regulating essential processes such as cell growth and migration are essential for cell homeostasis. As both GAGs and the lipid A disaccharide core of gram-negative bacteria contain negatively charged disaccharide units, we hypothesized that GAG-binding proteins could also recognize LPS and enclose cryptic antibiotic motifs. Here, we report novel antimicrobial peptides (AMPs) derived from heparin-binding proteins (HBPs), with specific activity against gram-negative bacteria and high LPS binding. We used computational tools to locate antimicrobial regions in 82% of HBPs, most of those colocalizing with putative heparin binding sites. To validate these results, we synthesized five candidates [HBP1-5] that showed remarkable activity against gram-negative bacteria, as well as a strong correlation between heparin and LPS binding. Structural characterization of these AMPs shows that heparin or LPS recognition promotes a conformational arrangement that favors binding. Among all analogs, HBP-5 displayed the highest affinity for both heparin and LPS, with antimicrobial activities against gram-negative bacteria at the nanomolar range. These results suggest that GAG-binding proteins are involved in LPS recognition, which allows them to act also as antimicrobial proteins. Some of the peptides reported here, particularly HBP-5, constitute a new class of AMPs with specific activity against gram-negative bacteria.

Foot-and-mouth disease virus (FMDV) poses a persistent threat to the livestock sector, urging a need for safer and more effective vaccines. As traditional control approaches relying on inactivated virus vaccines face limitations, exploring subunit vaccine strategies such as those based on synthetic peptides represents an attractive alternative, compliant with DIVA vaccine requirements. We previously reported that dendrimer structures combining virus-specific B- and T-cell epitopes-referred to as B2T-conferred solid protection against type-O FMDV in swine. More recently, we designed a synthetic strategy with broad application prospects, assembling peptides into a modular dendrimer platform named B2T-TB2, a dimeric version of the preceding construct, harboring up to six immunologically relevant epitopes. In this study, we demonstrate that a single low dose of this multiepitopic vaccine induces in swine a fast and robust neutralizing response covering a broad antigenic spectrum and confers full protection, portraying B2T-TB2 as a promising FMDV emergency vaccine.

This paper discusses the progress of antimicrobial peptide research from its early stages to its full blooming by the end of the 20th century. A consequential point throughout the discussion is that the availability of synthetic peptides -coinciding with the coming of age of solid phase peptide synthesis- was decisive for the remarkably fast development of the field.

The number of fungal infections is steadily increasing, with considerable morbidity and mortality. Additionally, antifungal resistance is a growing concern, highlighting the need to develop new treatment options. One alternative is the use of antimicrobial peptides (AMPs). The aim of this study was to assess the in vitro and in vivo antifungal activity of designed short AMPs, Act-6 and Act 8-20, derived from cecropin transcripts of beetles from the family Scarabaeidae, against eight reference strains of the pathogenic yeasts Candida and Cryptococcus. We also evaluated the effect of these modified AMPs on the biofilm, morphogenesis, and cell morphology of Candida albicans, as well as the in vivo activity via a murine model of disseminated candidiasis. The AMPs herein analyzed exhibit differential antifungal activity against the yeasts assessed, and inhibit biofilm, hyphae, and pseudohyphae formation with morphological alterations in C. albicans. Moreover, the fungal load in mice treated with these AMPs significantly decreased. Altogether, our results suggest that Act-6 and Act 8-20 are promising antifungal molecules to control mycoses.

The problems associated with the drugs currently used to treat leishmaniasis, including resistance, toxicity, and the high cost of some formulations, call for the urgent identification of new therapeutic agents with novel modes of action. The aggregated protein dye YAT2150 has been found to be a potent antileishmanial compound, with a half-maximal inhibitory concentration (IC50) of approximately 0.5 mu M against promastigote and amastigote stages of Leishmania infantum. The encapsulation in liposomes of YAT2150 significantly improved its in vitro IC50 to 0.37 and 0.19 mu M in promastigotes and amastigotes, respectively, and increased the half-maximal cytotoxic concentration in human umbilical vein endothelial cells to >50 mu M. YAT2150 became strongly fluorescent when binding intracellular protein deposits in Leishmania cells. This fluorescence pattern aligns with the proposed mode of action of this drug in the malaria parasite Plasmodium falciparum, the inhibition of protein aggregation. In Leishmania major, YAT2150 rapidly reduced ATP levels, suggesting an alternative antileishmanial mechanism. To the best of our knowledge, this first-in-class compound is the only one described so far having significant activity against both Plasmodium and Leishmania, thus being a potential drug for the treatment of co-infections of both parasites.

Activation of cannabinoid CB1 receptors (CB1R) by agonists induces analgesia but also induces cognitive impairment through the heteromer formed between CB1R and the serotonin 5HT2A receptor (5HT2AR). This side effect poses a serious drawback in the therapeutic use of cannabis for pain alleviation. Peptides designed from the transmembrane helices of CB1R, which are predicted to bind 5HT2AR and alter the stability of the CB1R-5HT2AR heteromer, have been shown to avert CB1R agonist-induced cognitive impairment while preserving analgesia. Using these peptides as templates, we have now designed nonpeptidic small molecules that prevent CB1R-5HT2AR heteromerization in bimolecular fluorescence complementation assays and the heteromerization-dependent allosteric modulations in cell signaling experiments. These results provide proof-of-principle for the design of optimized ligand-based disruptors of the CB1R-5HT2AR heteromer, opening new perspectives for in vivo studies.

Security and RAS are two non-functional requirements under focus for current systems developed for the computing continuum. Due to the increased number of interconnected computer systems across the continuum, security becomes especially pervasive at all levels, from the smallest edge device to the high-performance cloud at the other end. Similarly, RAS (Reliability, Availability, and Serviceability) ensures the robustness of a system towards hardware defects. Namely, making them reliable, with high availability and design for easy service. In this paper and as a result of the Vitamin-V Eu project, the authors detail the comprehensive approach to malware and hardware attack detection; as well as, the RAS features envisioned for future systems across the computing continuum.

Antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics in the face of ever-increasing resistance. However, many AMPs fail to progress into clinics due to unexpected difficulties found in preclinical in vivo phases. Our research has focused on crotalicidin (Ctn), an AMP from snake venom, and a fragment thereof, Ctn[15-34], with improved in vitro antimicrobial and anticancer activities and remarkable serum stability. As the retroenantio versions of both AMPs maintained favorable profiles, in this work, we evaluate the in vivo efficacy of both the native-sequence AMPs and their retroenantio counterparts in a murine infection model with Acinetobacter baumannii. A significant reduction in bacterial levels is found in the mice treated with Ctn[15-34]. However, contrary to expectations, the retroenantio analogs either exhibit toxicity or lack efficacy when administered to mice. Our findings underscore the critical importance of in vivo infection model evaluation to fully calibrate the therapeutic potential of AMPs.

Blood-brain barrier (BBB) peptide-shuttles (BBBpS) are able to translocate the BBB and reach the brain. Despite the importance of brain targeting in pharmacology, BBBpS are poorly characterized. Currently, their development relies on the empiric assumption that cell-penetrating peptides (CPPs), with proven ability to traverse lipid membranes, will likewise behave as a BBBpS. The relationship between CPPs/BBBpS remains elusive and, to the best of our knowledge, has not hitherto been subject to thorough experimental scrutiny. In this work, we have identified/quantified the main physicochemical properties of BBBpS and then searched for CPPs with these properties, hence potential BBBpS. The specific features found for BBBpS are: (i) small size, (ii) none or few aromatic residues, (iii) hydrophobic, and (iv) slight cationic nature. Then, we selected the 10 scoring best in an ordinary least squares analysis, and tested them in vitro and in vivo. Overall, we identified the molecular determinants for brain targeting by peptides, devised a methodology that can be used to assist in the design of peptides with potential brain penetration from amino acid residue sequences, and found four new BBBpS within the CPP library.

ABSTRACT The alarming rise of antibiotic-resistant bacterial infections is driving efforts to develop alternatives to conventional antibiotics. In this context, antimicrobial peptides (AMPs) have emerged as promising candidates for their ability to target a broad range of microorganisms. However, the development of AMPs with optimal potency, selectivity, and/or stability profiles remains a challenge. To address it, computational tools for predicting AMP properties and designing novel peptides have gained increasing attention. PyAMPA is a novel platform for AMP discovery. It consists of five modules, namely AMPScreen, AMPValidate, AMPSolve, AMPMutate, and AMPOptimize, that allow high-throughput proteome inspection, candidate screening, and optimization through point-mutation and genetic algorithms. The platform also offers additional tools for predicting and evaluating AMP properties, including antimicrobial and cytotoxic activity, and peptide half-life. By providing innovative and accessible inroads into AMP motifs in proteomes, PyAMPA will enable advances in AMP development and potential translation into clinically useful molecules. PyAMPA is available at: https://github.com/SysBioUAB/PyAMPA IMPORTANCE This paper introduces PyAMPA, a new bioinformatics platform designed for the discovery and optimization of antimicrobial peptides (AMPs). It addresses the urgent need for new antimicrobials due to the rise of antibiotic-resistant infections. PyAMPA, with its five predictive modules -AMPScreen, AMPValidate, AMPSolve, AMPMutate and AMPOptimize, enables high-throughput screening of proteomes to identify potential AMP motifs and optimize them for clinical use. Its unique approach, combining prediction, design, and optimization tools, makes PyAMPA a robust solution for developing new AMP-based therapies, offering a significant advance in combatting antibiotic resistance.