The Designed Pore-Forming Antimicrobial Peptide C14R Combines Excellent Activity against the Major Opportunistic Human Pathogen Pseudomonas aeruginosa with Low Cytotoxicity.

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10-15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future.

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.

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.

Identification of a Phage Display-Derived Peptide Interacting with the N-Terminal Region of Factor VII Activating Protease (FSAP) Enables Characterization of Zymogen Activation.

Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

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.

Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection.

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

Respiratory ß-2-Microglobulin exerts pH dependent antimicrobial activity.

The respiratory tract is a major entry site for microbial pathogens. To combat bacterial infections, the immune system has various defense mechanisms at its disposal, including antimicrobial peptides (AMPs). To search for novel AMPs from the respiratory tract, a peptide library from human broncho-alveolar-lavage (BAL) fluid was screened for antimicrobial activity by radial diffusion assays allowing the efficient detection of antibacterial activity within a small sample size. After repeated testing-cycles and subsequent purification, we identified ß-2-microglobulin (B2M) in antibacterially active fractions. B2M belongs to the MHC-1 receptor complex present at the surface of nucleated cells. It is known to inhibit the growth of Listeria monocytogenes and Escherichia coli and to facilitate phagocytosis of Staphylococcus aureus. Using commercially available B2M we confirmed a dose-dependent inhibition of Pseudomonas aeruginosa and L. monocytogenes. To characterize AMP activity within the B2M sequence, peptide fragments of the molecule were tested for antimicrobial activity. Activity could be localized to the C-terminal part of B2M. Investigating pH dependency of the antimicrobial activity of B2M demonstrated an increased activity at pH values of 5.5 and below, a hallmark of infection and inflammation. Sytox green uptake into bacterial cells following the exposure to B2M was determined and revealed a pH-dependent loss of bacterial membrane integrity. TEM analysis showed areas of disrupted bacterial membranes in L. monocytogenes incubated with B2M and high amounts of lysed bacterial cells. In conclusion, B2M as part of a ubiquitous cell surface complex may represent a potent antimicrobial agent by interfering with bacterial membrane integrity.

An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection.

SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID50 of virus stocks, antiviral efficiencies (IC50 values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research.

A Placenta Derived C-Terminal Fragment of ß-Hemoglobin With Combined Antibacterial and Antiviral Activity.

The placenta acts as physical and immunological barrier against the transmission of viruses and bacteria from mother to fetus. However, the specific mechanisms by which the placenta protects the developing fetus from viral and bacterial pathogens are poorly understood. To identify placental peptides and small proteins protecting from viral and bacterial infections, we generated a peptide library from 10 kg placenta by chromatographic means. Screening the resulting 250 fractions against Herpes-Simplex-Virus 2 (HSV-2), which is rarely transmitted through the placenta, in a cell-based system identified two adjacent fractions with significant antiviral activity. Further rounds of chromatographic purification and anti-HSV-2 testing allowed to purify the bioactive peptide. Mass spectrometry revealed the presence of a 36-mer derived from the C-terminal region of the hemoglobin ß subunit. The purified and corresponding chemically synthesized peptide, termed HBB(112-147), inhibited HSV-2 infection in a dose-dependent manner, with a mean IC50 in the median µg/ml range. Full-length hemoglobin tetramer had no antiviral activity. HBB(112-147) did not impair infectivity by direct targeting of the virions but prevented HSV-2 infection at the cell entry level. The peptide was inactive against Human Immunodeficiency Virus Type 1, Rubella and Zika virus infection, suggesting a specific anti-HSV-2 mechanism. Notably, HBB(112-147) has previously been identified as broad-spectrum antibacterial agent. It is abundant in placenta, reaching concentrations between 280 and 740 µg/ml, that are well sufficient to inhibit HSV-2 and prototype Gram-positive and -negative bacteria. We here additionally show, that HBB(112-147) also acts potently against Pseudomonas aeruginosa strains (including a multi-drug resistant strain) in a dose dependent manner, while full-length hemoglobin is inactive. Interestingly, the antibacterial activity of HBB(112-147) was increased under acidic conditions, a hallmark of infection and inflammatory conditions. Indeed, we found that HBB(112-147) is released from the hemoglobin precursor by Cathepsin D and Napsin A, acidic proteases highly expressed in placental and other tissues. We propose that upon viral or bacterial infection, the abundant hemoglobin precursor is proteolytically processed to release HBB(112-147), a broadly active antimicrobial innate immune defense peptide.

Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.

Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100 µm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150 µM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.