Cell Penetrating Peptide Sequence Research Articles

AAV-aMTD-Parkin, a therapeutic gene delivery cargo, enhances motor and cognitive functions in Parkinson's and Alzheimer’s diseases

Neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD), have a global prevalence and profoundly impact both motor and cognitive functions. Although adeno-associated virus (AAV)-based gene therapy has shown promise, its application for treating central nervous system (CNS) diseases faces several challenges, including effective delivery of AAV vectors across the blood-brain barrier, determining optimal dosages, and achieving targeted distribution. To address these challenges, we have developed a fusion delivery therapeutic cargo called AAV-aMTD-Parkin, which combines a hydrophobic cell-penetrating peptide sequence with the DNA sequences of AAV and Parkin. By employing this fusion delivery platform at lower dosages compared to zolgensma, we have achieved significant enhancements in cell and tissue permeability, while reducing the occurrence of common pathological protein aggregates. Consequently, motor and cognitive functions were restored in animal models of PD and AD. With its dual functionality in addressing PD and AD, AAV-aMTD-Parkin holds immense potential as a novel class of therapeutic biologics for prevalent CNS diseases.

TriplEP-CPP: Algorithm for Predicting the Properties of Peptide Sequences.

Advancements in medicine and pharmacology have led to the development of systems that deliver biologically active molecules inside cells, increasing drug concentrations at target sites. This improves effectiveness and duration of action and reduces side effects on healthy tissues. Cell-penetrating peptides (CPPs) show promise in this area. While traditional medicinal chemistry methods have been used to develop CPPs, machine learning techniques can speed up and reduce costs in the search for new peptides. A predictive algorithm based on machine learning models was created to identify novel CPP sequences using molecular descriptors using a combination of algorithms like k-nearest neighbors, gradient boosting, and random forest. Some potential CPPs were found and tested for cytotoxicity and penetrating ability. A new low-toxicity CPP was discovered from the Rhopilema esculentum venom proteome through this study.

Liposome nanoparticle conjugation and cell penetrating peptide sequences (CPPs) enhance the cellular delivery of the tau aggregation inhibitor RI-AG03.

Given the pathological role of Tau aggregation in Alzheimer's disease (AD), our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that the peptide can traverse the cell membrane. Here we examine the cellular uptake and intracellular trafficking of RI-AG03, in both a free and liposome-conjugated form. We also characterize the impact of adding the cell-penetrating peptide (CPP) sequences, polyarginine (polyR) or transactivator of transcription (TAT), to RI-AG03. Our data show that liposome conjugation of CPP containing RI-AG03 peptides, with either the polyR or TAT sequence, increased cellular liposome association three-fold. Inhibition of macropinocytosis modestly reduced the uptake of unconjugated and RI-AG03-polyR-linked liposomes, while having no effect on RI-AG03-TAT-conjugated liposome uptake. Further supporting macropinocytosis-mediated internalization, a 'fair' co-localisation of the free and liposome-conjugated RI-AG03-polyR peptide with macropinosomes and lysosomes was observed. Interestingly, we also demonstrate that RI-AG03-polyR detaches from liposomes following cellular uptake, thereby largely evading organellar entrapment. Collectively, our data indicate that direct membrane penetration and macropinocytosis are key routes for the internalization of liposomes conjugated with CPP containing RI-AG03. Our study also demonstrates that peptide-liposomes are suitable nanocarriers for the cellular delivery of RI-AG03, furthering their potential use in targeting Tau pathology in AD.

Flotillin-1 palmitoylation is essential for its stability and subsequent tumor promoting capabilities.

Flotillin-1 contributes to invasion and metastasis in triple negative breast cancer (TNBC) and is modified post-translationally through palmitoylation. Palmitoylation, the process of conjugating palmitoyl-CoA to proteins, plays an essential role in protein stability and trafficking. Thus far, there has not been any investigation into the role of flotillin-1 palmitoylation in the context of metastasis in vivo. To address the role of flotillin-1 palmitoylation in metastasis, MDA-MB-231 cells expressing palmitoylation defective flotillin-1 constructs were used as models. Compared to flotillin-1 WT expressing tumors, flotillin-1 palmitoylation defective displayed abrogated tumor progression and lung metastasis in vivo in both spontaneous and experimental models. Further mechanistic investigation led to the identification of zDHHC5 as the main palmitoyl acyltransferase responsible for palmitoylating endogenous flotillin-1. Modulation of flotillin-1 palmitoylation status through mutagenesis, zDHHC5 silencing, and 2-bromopalmitate inhibition all resulted in the proteasomal degradation of flotillin-1 protein. To assess if flotillin-1 palmitoylation can be inhibited for potential clinical relevance, we designed a competitive peptide fused to a cell penetrating peptide sequence, which displayed efficacy in blocking flotillin-1 palmitoylation in vitro without altering palmitoylation of other zDHHC5 substrates, highlighting its specificity. Additionally, TNBC xenograft tumor models expressing a doxycycline inducible flotillin-1 palmitoylation inhibiting peptide displayed attenuated tumor growth and lung metastasis. Collectively, these results reveal a novel palmitoylation dependent mechanism which is essential for the stability of flotillin-1 protein. More specifically, disruption of flotillin-1 palmitoylation through mutagenesis or competitive peptide promoted flotillin-1 protein degradation, subsequently impeding its tumor promoting and metastasis-inducing effects in TNBC tumor models.

Adamantylglycine as a high-affinity peptide label for membrane transport monitoring and regulation.

The non-canonical amino acid adamantylglycine (Ada) is introduced into peptides to allow high-affinity binding to cucurbit[7]uril (CB7). Introduction of Ada into a cell-penetrating peptide (CPP) sequence had minimal influence on the membrane transport, yet enabled up- and down-regulation of the membrane transport activity.

Design of peptide therapeutics as protein-protein interaction inhibitors to treat neurodegenerative diseases.

Peptide therapeutics are an emerging class of drugs to treat neurodegenerative diseases by inhibiting protein-protein interactions (PPIs). Nerinetide has recently emerged as a promising therapeutic for the treatment of ischemic stroke and Alzheimer's Disease (AD). The design of this potent neuroprotective agent includes a cell penetrating peptide sequence that achieves delivery into neurons and a protein-protein inhibitory sequence that achieves inhibition of protein complex formation through mimicry. In this study, we deconstruct the nerinetide sequence and study the relationship between plasma stability, intraneuronal delivery and drug efficacy to provide design guidelines for the development of next generation, peptidic PPI inhibitors to treat neurodegenerative diseases.

The light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibits cortical reaction in mouse oocytes.

Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction. Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes. Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis. Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes.

Sneaking in SpyCatcher using cell penetrating peptides for in vivo imaging

In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable ‘cargo’ , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.

Fabrication, characterization and evaluation of a new designed botulinum toxin-cell penetrating peptide nanoparticulate complex.

To have a better and longer effect, botulinum neurotoxin (BoNT) is injected several times in a treatment course, which could increase side effects and cost. Some of the most cutting-edge strategies being investigated for proteins to their physiologic targets involve the reformulation of BoNT based on peptide-based delivery systems. For this purpose, cell-penetrating peptides (CPPs) are of particular interest because of their capacity to cross the biological membranes. A short and simple CPP sequence was used as a carrier to create nanocomplex particles from BoNT/A, with the purpose of increasing toxin entrapment by target cells, reducing diffusion, and increasing the duration of the effect. CPP-BoNT/A nanocomplexes were formed by polyelectrolyte complex (PEC) method, considering the anionic structure of botulinum toxin and the cationic CPP sequence. The cellular toxicity, and absorption profile of the complex nanoparticles were evaluated, and the digit abduction score (DAS) was used to assess the local muscle weakening efficacy of BoNT/A and CPP-BoNT/A. The provided optimized polyelectrolyte complex nanoparticles had a 244 ± 20nm particle size and 0.28 ± 0.04 PdI. In cellular toxicity, CPP-BoNT/A nanocomplexes as extended-release formulations of BoNT/A showed that nanocomplexes had a more toxic effect than BoNT/A. Furthermore, the comparison of weakening effectiveness on muscle was done among nanoparticles and free toxin on mice based on the digit abduction score (DAS) method, and nanocomplexes had a slower onset effect and a longer duration of action than toxin. Using PEC method allowed us to form nanocomplex from proteins, and peptides without a covalent bond and harsh conditions. The muscle-weakening effect of toxin in CPP-BoNT/A nanocomplexes showed acceptable efficacy and extended-release pattern.

In Silico Screening and Optimization of Cell-Penetrating Peptides Using Deep Learning Methods

Cell-penetrating peptides (CPPs) have great potential to deliver bioactive agents into cells. Although there have been many recent advances in CPP-related research, it is still important to develop more efficient CPPs. The development of CPPs by in silico methods is a very useful addition to experimental methods, but in many cases it can lead to a large number of false-positive results. In this study, we developed a deep-learning-based CPP prediction method, AiCPP, to develop novel CPPs. AiCPP uses a large number of peptide sequences derived from human-reference proteins as a negative set to reduce false-positive predictions and adopts a method to learn small-length peptide sequence motifs that may have CPP tendencies. Using AiCPP, we found that short peptide sequences derived from amyloid precursor proteins are efficient new CPPs, and experimentally confirmed that these CPP sequences can be further optimized.

In situ quantitative measurements on MMP-9 activity in single living cells by single molecule fluorescence correlation spectroscopy.

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor progression. It is of great significance to establish a sensitive in situ assay strategy for MMP-9 activity in single living cells. Here a novel in situ single molecule spectroscopy method based on the fluorescence correlation spectroscopy (FCS) technique was proposed for measuring the MMP-9 activity at different locations within single living cells, using a fluorescent specific peptide and a reference dye as dual probes. The measurement principle is based on the decrease of the ratiometric translational diffusion time of dual probes in the detection volume due to the peptide cleavage caused by MMP-9. The peptide probe was designed to be composed of an MMP-9 cleavage and cell-penetrating peptide sequence that was labeled with a fluorophore and conjugated with a streptavidin (SAV) molecule. The ratiometric translational diffusion time was used as the measurement parameter to eliminate the effect of intracellular uncertain viscosity. The linear relationship between the ratiometric diffusion time and MMP-9 activity was established, and applied to the determination of enzymatic activity in cell lysates as well as the evaluation of the inhibitory effects of different inhibitors on MMP-9. More importantly, the method was successfully used to dynamically determine MMP-9 activity in single living cells or under the stimulation with phorbol 12-myristate 13-acetate (PMA) and inhibitors.

Recombinant cell-penetrating trichosanthin synergizes anti-PD-1 therapy in colorectal tumor.

Alleviating immunosuppression of the tumor microenvironment is an important strategy to improve immune checkpoint therapy. It is an urgent but unmet need to develop adjuvant therapeutics for assisting the mainstay immunotherapies. Trichosanthin is an approved gynecology drug in China and its immunomodulatory effects have drawn much attention as an old drug for new applications in cancer. In this work, a recombinant cell-penetrating trichosanthin (rTCS-LMWP) was prepared via genetic fusion of a cell-penetrating peptide sequence (LMWP) to trichosanthin aiming to overcome the intratumoral penetration and intracellular delivery challenges. The potential of trichosanthin as an adjuvant therapy was explored, including its effects on tumor cells, antigen-presenting cells, tumor immune microenvironment, and the synergistic effect in combination with anti-PD-1. The results revealed that rTCS-LMWP can stimulate the maturation of dendritic cells via activating the STING-TBK1-IRF3 pathway, repolarize the protumor M2-type macrophages, and upregulate the pro-inflammatory cytokine expression. Moreover, rTCS-LMWP can enhance anti-PD-1 therapeutic efficacy in a CT26-bearing mouse model. The synergistic effect involved the induction of immunogenic cell death in the tumors, the proliferation and functionalization of cytotoxic T cells, and the suppression of the immunosuppressive regulatory T cells. These findings indicate that trichosanthin can be developed as an immunomodulator to facilitate cancer immunotherapy.

Self-cleaving protein linkers with modulated pH-responsiveness: A new platform for selective control of protein drug function

In this study, we propose a self-cleaving protein that responds to acidic pH, pH inteinN150, as a pH-responsive linker for the selective delivery of protein-based drugs. Being stable at neutral and degradable at weakly acidic pH, pH inteinN150 can be obtained by mutating key amino acids of pH intein, thus stimulating self-cleavage. Unlike chemical linkers, which require additional conjugation steps, protein linkers can be incorporated into protein pharmaceuticals during protein expression. As proof-of-concepts, intracellular penetration of proteins can be selectively turned on or off by cleaving pH inteinN150 near the cell-penetrating peptide sequence at weakly acidic pH. Furthermore, the apoptosis-inducing activity of human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) can be selectively activated by cleaving pH inteinN150 adjacent to the albumin binding domain (ABD) at weakly acidic pH. Thus, we expect that this new protein linker can be used for actively controlling various protein-based drugs responding to delicate pH variations around inflammatory or cancerous tissues. These findings have been revealed in an in vivo tumor xenograft mouse model showing elongated systemic circulation and selective induction of tumor toxicity by ABD-pH inteinN150-hTRAIL.

In Silico Prediction of Anti-Infective and Cell-Penetrating Peptides from Thalassophryne nattereri Natterin Toxins.

The therapeutic potential of venom-derived peptides, such as bioactive peptides (BAPs), is determined by specificity, stability, and pharmacokinetics properties. BAPs, including anti-infective or antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs), share several physicochemical characteristics and are potential alternatives to antibiotic-based therapies and drug delivery systems, respectively. This study used in silico methods to predict AMPs and CPPs derived from natterins from the venomous fish Thalassophryne nattereri. Fifty-seven BAPs (19 AMPs, 8 CPPs, and 30 AMPs/CPPs) were identified using the web servers CAMP, AMPA, AmpGram, C2Pred, and CellPPD. The physicochemical properties were analyzed using ProtParam, PepCalc, and DispHred tools. The membrane-binding potential and cellular location of each peptide were analyzed using the Boman index by APD3, and TMHMM web servers. All CPPs and two AMPs showed high membrane-binding potential. Fifty-four peptides were located in the plasma membrane. Peptide immunogenicity, toxicity, allergenicity, and ADMET parameters were evaluated using several web servers. Sixteen antiviral peptides and 37 anticancer peptides were predicted using the web servers Meta-iAVP and ACPred. Secondary structures and helical wheel projections were predicted using the PEP-FOLD3 and Heliquest web servers. Fifteen peptides are potential lead compounds and were selected to be further synthesized and tested experimentally in vitro to validate the in silico screening. The use of computer-aided design for predicting peptide structure and activity is fast and cost-effective and facilitates the design of potent therapeutic peptides. The results demonstrate that toxins form a natural biotechnological platform in drug discovery, and the presence of CPP and AMP sequences in toxin families opens new possibilities in toxin biochemistry research.

Cell-Penetrating Peptides with Unexpected Anti-Amyloid Properties.

Cell-penetrating peptides (CPPs) with sequences derived originally from a prion protein (PrP) have been shown to exhibit both anti-prion and anti-amyloid properties particularly against prion proteins and the amyloid-β (Aβ) peptide active in Alzheimer’s disease. These disease-modifying properties are so far observed in cell cultures and in vitro. The CPP sequences are composed of a hydrophobic signal sequence followed by a highly positively charged hexapeptide segment. The original signal sequence of the prion protein can be changed to the signal sequence of the NCAM1 protein without losing the anti-prion activity. Although the detailed molecular mechanisms of these CPP peptides are not fully understood, they do form amyloid aggregates by themselves, and molecular interactions between the CPPs and PrP/Aβ can be observed in vitro using various spectroscopic techniques. These initial intermolecular interactions appear to re-direct the aggregation pathways for prion/amyloid formation to less cell-toxic molecular structures (i.e., co-aggregates), which likely is why the disease-inducing PrP/Aβ aggregation is counteracted in vivo.

Cell penetrating peptides coupled to an endothelial nitric oxide synthase sequence alter endothelial permeability

ABSTRACT Delivery of cargo to cells through the use of cell-penetrating peptide (CPP) sequences is an area of rich investigation for targeted therapeutics. Specific to the endothelium, the layer of cells that cover every blood vessel in the body, the loss or alteration of a key enzyme, endothelial nitric oxide synthase (eNOS), is known to contribute to endothelial health during severe, infectious challenge. While the beneficial effects of eNOS are often thought to be mediated through the generation of nitric oxide, some protection is theorized to be through eNOS binding to regulatory pathways via a pentabasic RRKRK motif. We hypothesized that delivery of the eNOS-RRKRK peptide sequence using common CPPs would allow protection against gram-negative lipopolysaccharide (LPS). Combination of the eNOS-RRKRK sequence to the CPP antennapedia (AP) reduced the impact of LPS-induced permeability in cultured human microvascular endothelial cells (HMVECs) as measured by transendothelial electrical resistance (TEER). There was also a modest reduction in cytokine production, however it was observed that AP alone significantly impaired LPS-induced endothelial permeability and cytokine production. In comparison, the CPP trans-activator of transcription (TAT) did not significantly alter endothelial inflammation by itself. When TAT was coupled to the eNOS-RRKRK sequence, protection against LPS-induced permeability was still demonstrated, however cytokine production was not reduced. These data demonstrate that the RRKRK sequence of eNOS can offer some NO-independent protection against LPS-mediated endothelial inflammation, however the degree of protection is highly dependent on the type of CPP utilized for cargo delivery.

Deep Learning Enables Discovery of a Short Nuclear Targeting Peptide for Efficient Delivery of Antisense Oligomers.

Therapeutic macromolecules such as proteins and oligonucleotides can be highly efficacious but are often limited to extracellular targets due to the cell’s impermeable membrane. Cell-penetrating peptides (CPPs) are able to deliver such macromolecules into cells, but limited structure–activity relationships and inconsistent literature reports make it difficult to design effective CPPs for a given cargo. For example, polyarginine motifs are common in CPPs, promoting cell uptake at the expense of systemic toxicity. Machine learning may be able to address this challenge by bridging gaps between experimental data in order to discern sequence–activity relationships that evade our intuition. Our earlier data set and deep learning model led to the design of miniproteins (>40 amino acids) for antisense delivery. Here, we leveraged and expanded our model with data augmentation in the short CPP sequence space of the data set to extrapolate and discover short, low-arginine-content CPPs that would be easier to synthesize and amenable to rapid conjugation to desired cargo, and with minimal in vivo toxicity. The lead predicted peptide, termed P6, is as active as a polyarginine CPP for the delivery of an antisense oligomer, while having only one arginine side chain and 18 total residues. We determined the pentalysine motif and the C-terminal cysteine of P6 to be the main drivers of activity. The antisense conjugate was able to enhance corrective splicing in an animal model to produce functional eGFP in heart tissue in vivo while remaining nontoxic up to a dose of 60 mg/kg. In addition, P6 was able to deliver an enzyme to the cytosol of cells. Our findings suggest that, given a data set of long CPPs, we can discover by extrapolation short, active sequences that deliver antisense oligomers.

Cell-Penetrating Peptides Predicted From CASC3, AKIP1, and AHRR Proteins.

Peptides can be used as research tools and for diagnostic or therapeutic applications. Peptides, alongside small molecules and antibodies, are used and are gaining further interest as protein-protein interaction (PPI) modulators. Peptides have high target specificity and high affinity, but, unlike small molecule modulators, they are not able to cross the cell membranes to reach their intracellular targets. To overcome this limitation, the special property of the cell-penetrating peptides (CPPs) could benefit their cause. CPPs are a class of peptides that can enter the cells and with them also deliver the attached cargoes. Today, with the advancement of in silico prediction tools and the availability of protein databases, designing new and multifunctional peptides that are able to reach intracellular targets and inhibit certain cellular processes in a very specific manner is reachable. Although there are several efficient CPP sequences already known, the discovery of new CPPs is crucial for the development of efficient delivery methods for both biotechnological and therapeutic applications. In this work, we chose 10 human nuclear proteins from which we predicted new potential CPP sequences by using three different CPP predictors: cell-penetrating peptide prediction tool, CellPPD, and SkipCPP-Pred. From each protein, one predicted CPP sequence was synthesized and its internalization into cells was assessed. Out of the tested sequences, three peptides displayed features characteristic to CPPs. These peptides and also the predicted peptide sequences could be used to design and modify new CPPs. In this work, we show that we can use protein sequences as input for generating new peptides with cell internalization properties. Three new CPPs, AHRR8-24, CASC3251-264, and AKIP127-37, can be further used for the delivery of other cargoes or designed into multifunctional peptides with capability of internalizing cells.

Generation of biologically active recombinant human OCT4 protein from E. coli.

The online version contains supplementary material available at 10.1007/s13205-021-02758-z.

Multistep optimization of a cell-penetrating peptide towards its antimicrobial activity.

Multidrug resistant (MDR) bacteria have adapted to most clinical antibiotics and are a growing threat to human health. One promising type of candidates for the everlasting demand of new antibiotic compounds constitute antimicrobial peptides (AMPs). These peptides act against different types of microbes by permeabilizing pathogen cell membranes, whereas being harmless to mammalian cells. Contrarily, another class of membrane-active peptides, namely cell-penetrating peptides (CPPs), is known to translocate in eukaryotic cells without substantially affecting the cell membrane. Since CPPs and AMPs share several physicochemical characteristics, we hypothesized if we can rationally direct the activity of a CPP towards antimicrobial activity. Herein, we describe the screening of a synthetic library, based on the CPP sC18, including structure-based design to identify the active residues within a CPP sequence and to discover novel AMPs with high activity. Peptides with increased hydrophobicity were tested against various bacterial strains, and hits were further optimized leading to four generations of peptides, with the last also comprising fluorinated amino acid building blocks. Interestingly, beside strong antibacterial activities, we also detected activity in cancer cells, while non-cancerous cells remained unharmed. The results highlight our new candidates, particularly those from generation 4, as a valuable and promising source for the development of future therapeutics with antibacterial activity and beyond.