Peptide Amphiphiles as Biodegradable Adjuvants for Efficient Retroviral Gene Delivery.

Retroviral gene delivery is the key technique for in vitro and ex vivo gene therapy. However, inefficient virion-cell attachment resulting in low gene transduction efficacy remains a major challenge in clinical applications. Adjuvants for ex vivo therapy settings need to increase transduction efficiency while being easily removed or degraded post-transduction to prevent the risk of venous embolism after infusing the transduced cells back to the bloodstream of patients, yet no such peptide system have been reported thus far. In this study, peptide amphiphiles (PAs) with a hydrophobic fatty acid and a hydrophilic peptide moiety that reveal enhanced viral transduction efficiency are introduced. The PAs form ß-sheet-rich fibrils that assemble into positively charged aggregates, promoting virus adhesion to the cell membrane. The block-type amphiphilic sequence arrangement in the PAs ensures efficient cell-virus interaction and biodegradability. Good biodegradability is observed for fibrils forming small aggregates and it is shown that via molecular dynamics simulations, the fibril-fibril interactions of PAs are governed by fibril surface hydrophobicity. These findings establish PAs as additives in retroviral gene transfer, rivalling commercially available transduction enhancers in efficiency and degradability with promising translational options in clinical gene therapy applications.

Development of N-Terminally Modified Variants of the CXCR4-Antagonistic Peptide EPI-X4 for Enhanced Plasma Stability.

EPI-X4, a natural peptide CXCR4 antagonist, shows potential for treating inflammation and cancer, but its short plasma stability limits its clinical application. We aimed to improve the plasma stability of EPI-X4 analogues without compromising CXCR4 antagonism. Our findings revealed that only the peptide N-terminus is prone to degradation. Consequently, incorporating d-amino acids or acetyl groups in this region enhanced peptide stability in plasma. Notably, EPI-X4 leads 5, 27, and 28 not only retained their CXCR4 binding and antagonism but also remained stable in plasma for over 8 h. Molecular dynamic simulations showed that these modified analogues bind similarly to CXCR4 as the original peptide. To further increase their systemic half-lives, we conjugated these stabilized analogues with large polymers and albumin binders. These advances highlight the potential of the optimized EPI-X4 analogues as promising CXCR4-targeted therapeutics and set the stage for more detailed preclinical assessments.

Conformational States of the CXCR4 Inhibitor Peptide EPI-X4-A Theoretical Analysis.

EPI-X4, an endogenous peptide inhibitor, has exhibited potential as a blocker of CXCR4-a G protein-coupled receptor. This unique inhibitor demonstrates the ability to impede HIV-1 infection and halt CXCR4-dependent processes such as tumor cell migration and invagination. Despite its promising effects, a comprehensive understanding of the interaction between EPI-X4 and CXCR4 under natural conditions remains elusive due to experimental limitations. To bridge this knowledge gap, a simulation approach was undertaken. Approximately 150,000 secondary structures of EPI-X4 were subjected to simulations to identify thermodynamically stable candidates. This simulation process harnessed a self-developed reactive force field operating within the ReaxFF framework. The application of the Two-Phase Thermodynamic methodology to ReaxFF facilitated the derivation of crucial thermodynamic attributes of the EPI-X4 conformers. To deepen insights, an ab initio density functional theory calculation method was employed to assess the electrostatic potentials of the most relevant (i.e., stable) EPI-X4 structures. This analytical endeavor aimed to enhance comprehension of the inhibitor's structural characteristics. As a result of these investigations, predictions were made regarding how EPI-X4 interacts with CXCR4. Two pivotal requirements emerged. Firstly, the spatial conformation of EPI-X4 must align effectively with the CXCR4 receptor protein. Secondly, the functional groups present on the surface of the inhibitor's structure must complement the corresponding features of CXCR4 to induce attraction between the two entities. These predictive outcomes were based on a meticulous analysis of the conformers, conducted in a gaseous environment. Ultimately, this rigorous exploration yielded a suitable EPI-X4 structure that fulfills the spatial and functional prerequisites for interacting with CXCR4, thus potentially shedding light on new avenues for therapeutic development.

Optimized peptide nanofibrils as efficient transduction enhancers for in vitro and ex vivo gene transfer.

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.

Peptide Bispecifics Inhibiting HIV-1 Infection by an Orthogonal Chemical and Supramolecular Strategy.

Viral infections pose a significant threat to human health, and effective antiviral strategies are urgently needed. Antiviral peptides have emerged as a promising class of therapeutic agents due to their unique properties and mechanisms of action. While effective on their own, combining antiviral peptides may allow us to enhance their potency and to prevent viral resistance. Here, we developed an orthogonal chemical strategy to prepare a heterodimeric peptide conjugate assembled on a protein-based nanoplatform. Specifically, we combined the optimized version of two peptides inhibiting HIV-1 by distinct mechanisms. Virus-inhibitory peptide (VIRIP) is a 20 amino acid fragment of α1-antitrypsin that inhibits HIV-1 by targeting the gp41 fusion peptide. Endogenous peptide inhibitor of CXCR4 (EPI-X4) is a 16-residue fragment of human serum albumin that prevents HIV-1 entry by binding to the viral CXCR4 co-receptor. Optimized forms of both peptides are assembled on supramolecular nanoplatforms through the streptavidin-biotin interaction. We show that the construct consisting of the two different peptides (SAv-VIR-102C9-EPI-X4 JM#173-C) shows increased activity against CCR5- and CXCR4-tropic HIV-1 variants. Our results are a proof of concept that peptides with different modes of action can be assembled on nanoplatforms to enhance their antiviral activity.

Data-mining unveils structure-property-activity correlation of viral infectivity enhancing self-assembling peptides.

Gene therapy via retroviral vectors holds great promise for treating a variety of serious diseases. It requires the use of additives to boost infectivity. Amyloid-like peptide nanofibers (PNFs) were shown to efficiently enhance retroviral gene transfer. However, the underlying mode of action of these peptides remains largely unknown. Data-mining is an efficient method to systematically study structure-function relationship and unveil patterns in a database. This data-mining study elucidates the multi-scale structure-property-activity relationship of transduction enhancing peptides for retroviral gene transfer. In contrast to previous reports, we find that not the amyloid fibrils themselves, but rather µm-sized ß-sheet rich aggregates enhance infectivity. Specifically, microscopic aggregation of ß-sheet rich amyloid structures with a hydrophobic surface pattern and positive surface charge are identified as key material properties. We validate the reliability of the amphiphilic sequence pattern and the general applicability of the key properties by rationally creating new active sequences and identifying short amyloidal peptides from various pathogenic and functional origin. Data-mining-even for small datasets-enables the development of new efficient retroviral transduction enhancers and provides important insights into the diverse bioactivity of the functional material class of amyloids.

ACE2-EGFR-MAPK signaling contributes to SARS-CoV-2 infection.

SARS-CoV-2 triggered the most severe pandemic of recent times. To enter into a host cell, SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2). However, subsequent studies indicated that other cell membrane receptors may act as virus-binding partners. Among these receptors, the epidermal growth factor receptor (EGFR) was hypothesized not only as a spike protein binder, but also to be activated in response to SARS-CoV-2. In our study, we aim at dissecting EGFR activation and its major downstream signaling pathway, the mitogen-activated signaling pathway (MAPK), in SARS-CoV-2 infection. Here, we demonstrate the activation of EGFR-MAPK signaling axis by the SARS-CoV-2 spike protein and we identify a yet unknown cross talk between ACE2 and EGFR that regulated ACE2 abundance and EGFR activation and subcellular localization, respectively. By inhibiting the EGFR-MAPK activation, we observe a reduced infection with either spike-pseudotyped particles or authentic SARS-CoV-2, thus indicating that EGFR serves as a cofactor and the activation of EGFR-MAPK contributes to SARS-CoV-2 infection.

Inverse design of viral infectivity-enhancing peptide fibrils from continuous protein-vector embeddings.

Amyloid-like nanofibers from self-assembling peptides can promote viral gene transfer for therapeutic applications. Traditionally, new sequences are discovered either from screening large libraries or by creating derivatives of known active peptides. However, the discovery of de novo peptides, which are sequence-wise not related to any known active peptides, is limited by the difficulty to rationally predict structure-activity relationships because their activities typically have multi-scale and multi-parameter dependencies. Here, we used a small library of 163 peptides as a training set to predict de novo sequences for viral infectivity enhancement using a machine learning (ML) approach based on natural language processing. Specifically, we trained an ML model using continuous vector representations of the peptides, which were previously shown to retain relevant information embedded in the sequences. We used the trained ML model to sample the sequence space of peptides with 6 amino acids to identify promising candidates. These 6-mers were then further screened for charge and aggregation propensity. The resulting 16 new 6-mers were tested and found to be active with a 25% hit rate. Strikingly, these de novo sequences are the shortest active peptides for infectivity enhancement reported so far and show no sequence relation to the training set. Moreover, by screening the sequence space, we discovered the first hydrophobic peptide fibrils with a moderately negative surface charge that can enhance infectivity. Hence, this ML strategy is a time- and cost-efficient way for expanding the sequence space of short functional self-assembling peptides exemplified for therapeutic viral gene delivery.

Development of a New Class of CXCR4-Targeting Radioligands Based on the Endogenous Antagonist EPI-X4 for Oncological Applications.

The peptide fragment of human serum albumin that was identified as an inhibitor of C-X-C motif chemokine receptor 4 (CXCR4), termed EPI-X4, was investigated as a scaffold for the development of CXCR4-targeting radio-theragnostics. Derivatives of its truncated version JM#21 (ILRWSRKLPCVS) were conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and tested in Jurkat and Ghost-CXCR4 cells. Ligand-1, -2, -5, -6, -7, -8, and -9 were selected for radiolabeling. Molecular modeling indicated that 177Lu-DOTA incorporation C-terminally did not interfere with the CXCR4 binding. Lipophilicity, in vitro plasma stability, and cellular uptake hinted 177Lu-7 as superior. In Jurkat xenografts, all radioligands showed >90% washout from the body within an hour, with the exception of 177Lu-7 and 177Lu-9. 177Lu-7 demonstrated best CXCR4-tumor targeting. Ex vivo biodistribution and single-photon emission computed tomography (SPECT)/positron emission tomography (PET)/CT imaging of 177Lu-7/68Ga-7 showed the same distribution profile for both radioligands, characterized by very low uptake in all nontargeted organs except the kidneys. The data support the feasibility of CXCR4-targeting with EPI-X4-based radioligands and designate ligand-7 as a lead candidate for further optimization.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Advanced EPI-X4 Derivatives Covalently Bind Human Serum Albumin Resulting in Prolonged Plasma Stability.

Advanced derivatives of the Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) have shown therapeutic efficacy upon topical administration in animal models of asthma and dermatitis. Here, we studied the plasma stability of the EPI-X4 lead compounds WSC02 and JM#21, using mass spectrometry to monitor the chemical integrity of the peptides and a functional fluorescence-based assay to determine peptide function in a CXCR4-antibody competition assay. Although mass spectrometry revealed very rapid disappearance of both peptides in human plasma within seconds, the functional assay revealed a significantly higher half-life of 9 min for EPI-X4 WSC02 and 6 min for EPI-X4 JM#21. Further analyses demonstrated that EPI-X4 WSC02 and EPI-X4 JM#21 interact with low molecular weight plasma components and serum albumin. Albumin binding is mediated by the formation of a disulfide bridge between Cys10 in the EPI-X4 peptides and Cys34 in albumin. These covalently linked albumin-peptide complexes have a higher stability in plasma as compared with the non-bound peptides and retain the ability to bind and antagonize CXCR4. Remarkably, chemically synthesized albumin-EPI-X4 conjugates coupled by non-breakable bonds have a drastically increased plasma stability of over 2 h. Thus, covalent coupling of EPI-X4 to albumin in vitro before administration or in vivo post administration may significantly increase the pharmacokinetic properties of this new class of CXCR4 antagonists.

PPI-Affinity: A Web Tool for the Prediction and Optimization of Protein-Peptide and Protein-Protein Binding Affinity.

Virtual screening of protein-protein and protein-peptide interactions is a challenging task that directly impacts the processes of hit identification and hit-to-lead optimization in drug design projects involving peptide-based pharmaceuticals. Although several screening tools designed to predict the binding affinity of protein-protein complexes have been proposed, methods specifically developed to predict protein-peptide binding affinity are comparatively scarce. Frequently, predictors trained to score the affinity of small molecules are used for peptides indistinctively, despite the larger complexity and heterogeneity of interactions rendered by peptide binders. To address this issue, we introduce PPI-Affinity, a tool that leverages support vector machine (SVM) predictors of binding affinity to screen datasets of protein-protein and protein-peptide complexes, as well as to generate and rank mutants of a given structure. The performance of the SVM models was assessed on four benchmark datasets, which include protein-protein and protein-peptide binding affinity data. In addition, we evaluated our model on a set of mutants of EPI-X4, an endogenous peptide inhibitor of the chemokine receptor CXCR4, and on complexes of the serine proteases HTRA1 and HTRA3 with peptides. PPI-Affinity is freely accessible at https://protdcal.zmb.uni-due.de/PPIAffinity.

In vitro Inhibition of HIV-1 by Cyclotide-Enriched Extracts of Viola tricolor.

Since viral infectious diseases continue to be a global health threat, new antiviral drugs are urgently needed. A unique class of therapeutic compounds are antimicrobial peptides (AMPs). They can be found in humans, bacteria and plants. Plants express a wide variety of such defense peptides as part of their innate immune system to protect from invading pathogens. Cyclotides are non-classical AMPs that share a similar structure. Their unique topology consists of a circular peptide backbone and disulfide bonds. In previous studies they have been attributed to a wide range of biological activities. To identify novel cyclotides with antiviral activity, we established a library of plant extracts largely consisting of cyclotide-rich species and screened them as inhibitors of HIV-1 infection. Subsequent extraction and fractionation revealed four cyclotide-containing subfractions from Viola tricolor with antiviral activity. These subfractions inhibited HIV-1 infection with IC50 values between 0.6 and 11.2 µg/ml, and selectivity indices of up to 8.1. The identification and characterization of antiviral cyclotides and the determination of the antiviral mechanisms may allow to develop novel agents to combat viral infections. Therefore, cyclotides represent a natural source of bioactive molecules with prospects for development as therapeutics.

Structure-Based Design of High-Affinity and Selective Peptidomimetic Hepsin Inhibitors.

In many solid tumors, increased upregulation of transmembrane serine proteases (TTSPs) leads to an overactivation of growth factors, which promotes tumor progression. Here, we have used a combinatorial methodology to develop high-affinity tetrapeptidic inhibitors. A previous virtual screening of 8000 peptide combinations against the crystal structure of the TTSP hepsin identified a series of recognition sequences, customized for the non-prime substrate binding (P) sites of this serine protease. A combination of the top recognition sequences with an electrophilic warhead resulted in highly potent inhibitors with good selectivity against coagulation proteases factor Xa and thrombin. Structure-activity relationships of two selected compounds were further elucidated by investigation of their stability in biological fluids as well as the influence of the warhead and truncated inhibitors on the inhibitory potency. Overall, this methodology yielded compounds as selective inhibitors for potential cancer drug development, where hepsin is overexpressed.

Endogenous Peptide Inhibitors of HIV Entry.

The discovery of the G-protein coupled-receptor (GPCR) CXCR4 as a major coreceptor of HIV-1 entry about three decades ago explained why the chemokine SDF-1/CXCL12 inhibits specific viral strains. The knowledge that RANTES, MlP-1α, and MlP-1ß specifically inhibit other primary HIV-1 strains allowed the rapid discovery of CCR5 as second major viral coreceptor and explained why individuals with deletions in CCR5 are protected against sexual HIV-1 transmission. Here, we provide an update on endogenous ligands of GPCRs that act as endogenous inhibitors of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) entry. In addition, we summarize the development of optimized derivatives of endogenous GPCR ligands and their perspectives as antiviral agents and beyond. Finally, we provide examples for other endogenous peptides that may contribute to our innate immune defense against HIV-1 and other viral pathogens and offer prospects for preventive or therapeutic development.

Dimerization of the Peptide CXCR4-Antagonist on Macromolecular and Supramolecular Protraction Arms Affords Increased Potency and Enhanced Plasma Stability.

Peptides are prime drug candidates due to their high specificity of action but are disadvantaged by low proteolytic stability. Here, we focus on the development of stabilized analogues of EPI-X4, an endogenous peptide antagonist of CXCR4. We synthesized macromolecular peptide conjugates and performed side-by-side comparison with their albumin-binding counterparts and considered monovalent conjugates, divalent telechelic conjugates, and Y-shaped peptide dimers. All constructs were tested for competition with the CXCR4 antibody-receptor engagement, inhibition of receptor activation, and inhibition of the CXCR4-tropic human immunodeficiency virus infection. We found that the Y-shaped conjugates were more potent than the parent peptide and at the same time more stable in human plasma, with a favorable outlook for translational studies.

An optimized derivative of an endogenous CXCR4 antagonist prevents atopic dermatitis and airway inflammation.

Aberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes such as cancer and inflammatory diseases. A natural fragment of serum albumin, named EPI-X4, has previously been identified as endogenous peptide antagonist and inverse agonist of CXCR4 and is a promising compound for the development of improved analogues for the therapy of CXCR4-associated diseases. To generate optimized EPI-X4 derivatives we here performed molecular docking analysis to identify key interaction motifs of EPI-X4/CXCR4. Subsequent rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4. The EPI-X4 derivative JM#21 bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. EPI-X4 JM#21 did not exert toxic effects in zebrafish embryos and suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. Moreover, topical administration of the optimized EPI-X4 derivative efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Thus, rationally designed EPI-X4 JM#21 is a novel potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted.

Computational modeling and experimental validation of the EPI-X4/CXCR4 complex allows rational design of small peptide antagonists.

EPI-X4, a 16-mer fragment of albumin, is a specific endogenous antagonist and inverse agonist of the CXC-motif-chemokine receptor 4 (CXCR4) and thus a key regulator of CXCR4 function. Accordingly, activity-optimized synthetic derivatives of EPI-X4 are promising leads for the therapy of CXCR4-linked disorders such as cancer or inflammatory diseases. We investigated the binding of EPI-X4 to CXCR4, which so far remained unclear, by means of biomolecular simulations combined with experimental mutagenesis and activity studies. We found that EPI-X4 interacts through its N-terminal residues with CXCR4 and identified its key interaction motifs, explaining receptor antagonization. Using this model, we developed shortened EPI-X4 derivatives (7-mers) with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors. Our work reveals the molecular details and mechanism by which the first endogenous peptide antagonist of CXCR4 interacts with its receptor and provides a foundation for the rational design of improved EPI-X4 derivatives.

Zika virus replication in glioblastoma cells: electron microscopic tomography shows 3D arrangement of endoplasmic reticulum, replication organelles, and viral ribonucleoproteins.

Structural changes of two patient-derived glioblastoma cell lines after Zika virus infection were investigated using scanning transmission electron tomography on high-pressure-frozen, freeze-substituted samples. In Zika-virus-infected cells, Golgi structures were barely visible under an electron microscope, and viral factories appeared. The cytosol outside of the viral factories resembled the cytosol of uninfected cells. The viral factories contained largely deranged endoplasmic reticulum (ER), filled with many so-called replication organelles consisting of a luminal vesicle surrounded by the ER membrane. Viral capsids were observed in the vicinity of the replication organelles (cell line #12537 GB) or in ER cisternae at large distance from the replication organelles (cell line #15747 GB). Near the replication organelles, we observed many about 100-nm-long filaments that may represent viral ribonucleoprotein complexes (RNPs), which consist of the RNA genome and N protein oligomers. In addition, we compared Zika-virus-infected cells with cells infected with a phlebovirus (sandfly fever Turkey virus). Zika virions are formed in the ER, whereas phlebovirus virions are assembled in the Golgi apparatus. Our findings will help to understand the replication cycle in the virus factories and the building of the replication organelles in glioblastoma cells.

A Fibrinogen Alpha Fragment Mitigates Chemotherapy-Induced MLL Rearrangements.

Rearrangements in the Mixed Lineage Leukemia breakpoint cluster region (MLLbcr) are frequently involved in therapy-induced leukemia, a severe side effect of anti-cancer therapies. Previous work unraveled Endonuclease G as the critical nuclease causing initial breakage in the MLLbcr in response to different types of chemotherapeutic treatment. To identify peptides protecting against therapy-induced leukemia, we screened a hemofiltrate-derived peptide library by use of an enhanced green fluorescent protein (EGFP)-based chromosomal reporter of MLLbcr rearrangements. Chromatographic purification of one active fraction and subsequent mass spectrometry allowed to isolate a C-terminal 27-mer of fibrinogen α encompassing amino acids 603 to 629. The chemically synthesized peptide, termed Fα27, inhibited MLLbcr rearrangements in immortalized hematopoietic cells following treatment with the cytostatics etoposide or doxorubicin. We also provide evidence for protection of primary human hematopoietic stem and progenitor cells from therapy-induced MLLbcr breakage. Of note, fibrinogen has been described to activate toll-like receptor 4 (TLR4). Dissecting the Fα27 mode-of action revealed association of the peptide with TLR4 in an antagonistic fashion affecting downstream NFκB signaling and pro-inflammatory cytokine production. In conclusion, we identified a hemofiltrate-derived peptide inhibitor of the genome destabilizing events causing secondary leukemia in patients undergoing chemotherapy.