Tryptophan- and arginine-rich antimicrobial peptides: Anti-infectives with great potential.

With the increasing prevalence of multidrug resistant (MDR) bacteria, there is a need to design synthetic antimicrobial peptides (AMPs) that are effective and selective for bacteria, i.e. non-toxic to mammalian cells. One design strategy, namely the use of tryptophan- and arginine-rich AMPs, is rooted in the study of natural AMPs that are composed mainly of these amino acids, such as lactoferricin, tritrpticin, and puroindoline. A number of important studies on these AMPs by the Vogel group are reviewed here. More recent work on W-/R-rich peptides is also presented. The examples show that these peptides represent anti-infectives with great potential.

Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design.

Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens.

Host defense peptides (HDPs) represent an alternative way to address the emergence of antibiotic resistance. Crocodylians are interesting species for the study of these molecules because of their potent immune system, which confers high resistance to infection. Profile hidden Markov models were used to screen the genomes of four crocodylian species for encoded cathelicidins and eighteen novel sequences were identified. Synthetic cathelicidins showed broad spectrum antimicrobial and antibiofilm activity against several clinically important antibiotic-resistant bacteria. In particular, the As-CATH8 cathelicidin showed potent in vitro activity profiles similar to the last-resort antibiotics vancomycin and polymyxin B. In addition, As-CATH8 demonstrated rapid killing of planktonic and biofilm cells, which correlated with its ability to cause cytoplasmic membrane depolarization and permeabilization as well as binding to DNA. As-CATH8 displayed greater antibiofilm activity than the human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus in a human organoid model of biofilm skin infection. Furthermore, As-CATH8 demonstrated strong antibacterial effects in a murine abscess model of high-density bacterial infections against clinical isolates of S. aureus and Acinetobacter baumannii, two of the most common bacterial species causing skin infections globally. Overall, this work expands the repertoire of cathelicidin peptides known in crocodylians, including one with considerable therapeutic promise for treating common skin infections.

Studying the Interactions of U24 from HHV-6 in Order to Further Elucidate Its Potential Role in MS.

A number of studies have suggested that human herpesvirus 6A (HHV-6A) may play a role in multiple sclerosis (MS). Three possible hypotheses have been investigated: (1) U24 from HHV-6A (U24-6A) mimics myelin basic protein (MBP) through analogous phosphorylation and interaction with Fyn-SH3; (2) U24-6A affects endocytic recycling by binding human neural precursor cell (NPC) expressed developmentally down-regulated protein 4-like WW3* domain (hNedd4L-WW3*); and (3) MS patients who express Killer Cell Immunoglobulin Like Receptor 2DL2 (KIR2DL2) on natural killer (NK) cells are more susceptible to HHV-6 infection. In this contribution, we examined the validity of these propositions by investigating the interactions of U24 from HHV-6B (U24-6B), a variant less commonly linked to MS, with Fyn-SH3 and hNedd4L-WW3* using heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) titrations and isothermal titration calorimetry (ITC). In addition, the importance of phosphorylation and the specific role of U24 in NK cell activation in MS patients were examined. Overall, the findings allowed us to shed light into the models linking HHV-6 to MS and the involvement of U24.

Viral Proteins with PxxP and PY Motifs May Play a Role in Multiple Sclerosis.

Multiple sclerosis (MS) is a debilitating disease that arises from immune system attacks to the protective myelin sheath that covers nerve fibers and ensures optimal communication between brain and body. Although the cause of MS is unknown, a number of factors, which include viruses, have been identified as increasing the risk of displaying MS symptoms. Specifically, the ubiquitous and highly prevalent Epstein-Barr virus, human herpesvirus 6, cytomegalovirus, varicella-zoster virus, and other viruses have been identified as potential triggering agents. In this review, we examine the specific role of proline-rich proteins encoded by these viruses and their potential role in MS at a molecular level.

Multifunctional Antibiotic-Host Defense Peptide Conjugate Kills Bacteria, Eradicates Biofilms, and Modulates the Innate Immune Response.

Effective anti-infective therapies are required to offset the rise in antibiotic resistance. A novel vancomycin-innate defense regulator conjugate (V-IDR1018) was constructed with multimodal functionality, including bacterial killing, biofilm eradication, and immune modulation. The conjugate killed bacteria within 30 min, exhibited potent activity against persister cells, and showed no susceptibility to antimicrobial resistance in tissue culture assays. Additionally, it stimulated the release of chemokine MCP-1 and anti-inflammatory cytokine IL-10 and suppressed pro-inflammatory IL-1ß from lipopolysaccharide-stimulated white blood cells. The conjugate demonstrated ∼90% eradication efficacy when assessed against the MRSA biofilm formed on an organoid human skin equivalent. Similarly, when evaluated in a murine, high-density skin abscess infection model using MRSA or Staphylococcus epidermidis, the conjugate decreased dermonecrosis and reduced bacterial load. The exceptional in vitro and in vivo efficacy of the conjugate, in addition to its safety profile, makes it a valuable candidate to treat complex infectious diseases.

Enzymatically releasable polyethylene glycol - host defense peptide conjugates with improved activity and biocompatibility.

Host defense peptides (HDPs) have been the subject of great interest for the treatment of multidrug-resistant bacterial infections due to their multimodal activity and low induction of resistance. However, aggregation, toxicity, and short biological half-life have limited their applicability for clinical treatment. Many methods have been explored to alleviate these issues, such as polymer (e.g., polyethylene glycol (PEG)) conjugation, but these are often accompanied by reductions in the activity of the HDP. Here, we detail the design of a novel PEG-HDP conjugate incorporating an enzymatic cleavage sequence targeting matrix metalloproteinases (MMPs) that accumulate at sites of inflammation and infection. Addition of the cleavage sequence onto either the N- or the C-terminal region of the parent peptide (peptide 73, a derivative of the HDP aurein 2.2) was explored to determine the location for optimal antimicrobial activity following MMP cleavage; furthermore, the susceptibility of the peptide to MMP cleavage after conjugation to 2 kDa or 5 kDa PEG was examined. The top candidate, L73, utilized an N-terminal cleavage site that was subsequently conjugated to a 2 kDa PEG polymer. Both L73 and the conjugate exhibited no antimicrobial activity in vitro until cleaved by purified MMP, which liberated a peptide fragment with 16- or 63-fold improved activity, respectively, corresponding to a minimum inhibitory concentration (MIC) of 8 µg/mL, comparable to that of peptide 73 (4 µg/mL). Furthermore, PEG conjugation improved the blood compatibility and reduced the aggregation tendency of the HDP in vitro, indicating enhanced biocompatibility. When administered as a single subcutaneous dose (~3.6 mg, or a peptide concentration of 142 mg/kg) in a mouse abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, the conjugate displayed strong activity, reducing abscess size and bacterial load by 73.3% and 58-fold, respectively. This activity was completely lost when the cleavage site was rendered resistant to MMPs by the substitution of two d-amino acids, supporting the hypothesis that antimicrobial activity was dependent on cleavage by MMPs, which were shown here to increasingly accumulate at the abscess site up to 18 h post infection. Finally, the conjugate displayed biocompatibility in vivo, with no identifiable toxicity or aggregation.

Host Defense Peptides: Dual Antimicrobial and Immunomodulatory Action.

The rapid rise of multidrug-resistant (MDR) bacteria has once again caused bacterial infections to become a global health concern. Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), offer a viable solution to these pathogens due to their diverse mechanisms of actions, which include direct killing as well as immunomodulatory properties (e.g., anti-inflammatory activity). HDPs may hence provide a more robust treatment of bacterial infections. In this review, the advent of and the mechanisms that lead to antibiotic resistance will be described. HDP mechanisms of antibacterial and immunomodulatory action will be presented, with specific examples of how the HDP aurein 2.2 and a few of its derivatives, namely peptide 73 and cG4L73, function. Finally, resistance that may arise from a broader use of HDPs in a clinical setting and methods to improve biocompatibility will be briefly discussed.

Lipidation of Antimicrobial Peptides as a Design Strategy for Future Alternatives to Antibiotics.

Multi-drug-resistant bacteria are becoming more prevalent, and treating these bacteria is becoming a global concern. One alternative approach to combat bacterial resistance is to use antimicrobial (AMPs) or host-defense peptides (HDPs) because they possess broad-spectrum activity, function in a variety of ways, and lead to minimal resistance. However, the therapeutic efficacy of HDPs is limited by a number of factors, including systemic toxicity, rapid degradation, and low bioavailability. One approach to circumvent these issues is to use lipidation, i.e., the attachment of one or more fatty acid chains to the amine groups of the N-terminus or a lysine residue of an HDP. In this review, we examined lipidated analogs of 66 different HDPs reported in the literature to determine: (i) whether there is a link between acyl chain length and antibacterial activity; (ii) whether the charge and (iii) the hydrophobicity of the HDP play a role; and (iv) whether acyl chain length and toxicity are related. Overall, the analysis suggests that lipidated HDPs with improved activity over the nonlipidated counterpart had acyl chain lengths of 8-12 carbons. Moreover, active lipidated peptides attached to short HDPs tended to have longer acyl chain lengths. Neither the charge of the parent HDP nor the percent hydrophobicity of the peptide had an apparent significant impact on the antibacterial activity. Finally, the relationship between acyl chain length and toxicity was difficult to determine due to the fact that toxicity is quantified in different ways. The impact of these trends, as well as combined strategies such as the incorporation of d- and non-natural amino acids or alternative approaches, will be discussed in light of how lipidation may play a role in the future development of antimicrobial peptide-based alternatives to current therapeutics.

Design, Engineering and Discovery of Novel α-Helical and ß-Boomerang Antimicrobial Peptides against Drug Resistant Bacteria.

In an era where the pipeline of new antibiotic development is drying up, the continuous rise of multi-drug resistant (MDR) and extensively drug resistant (XDR) bacteria are genuine threats to human health. Although antimicrobial peptides (AMPs) may serve as promising leads against drug resistant bacteria, only a few AMPs are in advanced clinical trials. The limitations of AMPs, namely their low in vivo activity, toxicity, and poor bioavailability, need to be addressed. Here, we review engineering of frog derived short α-helical AMPs (aurein, temporins) and lipopolysaccharide (LPS) binding designed ß-boomerang AMPs for further development. The discovery of novel cell selective AMPs from the human proprotein convertase furin is also discussed.

Cyclic Derivative of Host-Defense Peptide IDR-1018 Improves Proteolytic Stability, Suppresses Inflammation, and Enhances In Vivo Activity.

Host-defense peptides have drawn significant attention as new drugs or drug adjuvants to combat multidrug-resistant bacteria. In this study, we report the development of cyclic derivatives of the immunomodulatory and antibiofilm innate defense regulator peptide (IDR)-1018 based on three different synthetic strategies including head-to-tail cyclization (C1), side-chain-to-tail cyclization (C2), and a disulfide bond cross-linkage (C3). The generated mimetics showed enhanced proteolytic stability and reduced aggregation in vitro and in vivo. The C2 derivative exhibited exceptional ability to suppress inflammation and significantly reduce bacterial loads in a high-density Staphylococcus aureus murine skin infection model. The findings describe different routes to the creation of enzymatically stable mimetics of IDR-1018 and identify a promising new cyclic analogue against bacterial infections.

Towards Robust Delivery of Antimicrobial Peptides to Combat Bacterial Resistance.

Antimicrobial peptides (AMPs), otherwise known as host defence peptides (HDPs), are naturally occurring biomolecules expressed by a large array of species across the phylogenetic kingdoms. They have great potential to combat microbial infections by directly killing or inhibiting bacterial activity and/or by modulating the immune response of the host. Due to their multimodal properties, broad spectrum activity, and minimal resistance generation, these peptides have emerged as a promising response to the rapidly concerning problem of multidrug resistance (MDR). However, their therapeutic efficacy is limited by a number of factors, including rapid degradation, systemic toxicity, and low bioavailability. As such, many strategies have been developed to mitigate these limitations, such as peptide modification and delivery vehicle conjugation/encapsulation. Oftentimes, however, particularly in the case of the latter, this can hinder the activity of the parent AMP. Here, we review current delivery strategies used for AMP formulation, focusing on methodologies utilized for targeted infection site release of AMPs. This specificity unites the improved biocompatibility of the delivery vehicle with the unhindered activity of the free AMP, providing a promising means to effectively translate AMP therapy into clinical practice.

Insights into the mechanism of action of two analogues of aurein 2.2.

The naturally occurring host defense peptide (HDP), aurein 2.2, secreted by the amphibian Litoria aurea, acts as a moderate antibacterial, affecting Gram positive bacteria such as Staphylococcus aureus by forming selective ion pores. In a quest to find more active analogues of aurein 2.2, peptides 73 and 77 were discovered. These peptides were rich in arginine and tryptophan and found to have MICs of 4 µg/mL. Here we examined what impact the increased charge from +2 to +3 and a slight increase in hydrophobic moment relative to aurein 2.2 had on the mechanism of action of these two analogues. Using a time-kill assay, both peptides 73 and 77 were found to kill bacteria more effectively than the parent peptide. Using solution CD and NMR, the peptides were found to not adopt a continuous α-helical structure, i.e. the analogues were not helical from residue 1-13 like the parent peptide. Results obtained from oriented CD (OCD), DiSC35 and pyranine assays and a gel retardation experiment showed that the peptides did not function by membrane perturbation and further showed that peptide 73 and 77 did not interact with DNA. Overall, the data were consistent with these peptides acting as cell penetrating peptides with intracellular targets, which did not appear to be DNA.

Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules.

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

Reassessing the Host Defense Peptide Landscape.

Current research has demonstrated that small cationic amphipathic peptides have strong potential not only as antimicrobials, but also as antibiofilm agents, immune modulators, and anti-inflammatories. Although traditionally termed antimicrobial peptides (AMPs) these additional roles have prompted a shift in terminology to use the broader term host defense peptides (HDPs) to capture the multi-functional nature of these molecules. In this review, we critically examined the role of AMPs and HDPs in infectious diseases and inflammation. It is generally accepted that HDPs are multi-faceted mediators of a wide range of biological processes, with individual activities dependent on their polypeptide sequence. In this context, we explore the concept of chemical space as it applies to HDPs and hypothesize that the various functions and activities of this class of molecule exist on independent but overlapping activity landscapes. Finally, we outline several emerging functions and roles of HDPs and highlight how an improved understanding of these processes can potentially be leveraged to more fully realize the therapeutic promise of HDPs.

Aurein-Derived Antimicrobial Peptides Formulated with Pegylated Phospholipid Micelles to Target Methicillin-Resistant Staphylococcus aureus Skin Infections.

Antimicrobial peptides have been the focus of considerable research; however, issues associated with toxicity and aggregation have the potential to limit clinical applications. Here, a derivative of a truncated version of aurein 2.2 (aurein 2.2Δ3), namely peptide 73, was investigated, along with its d-amino acid counterpart (D-73) and a retro-inverso version (RI-73). A version that incorporated a cysteine residue to the C-terminus (73c) was also generated, as this form is required to covalently attach antimicrobial peptides to polymers (e.g., polyethylene glycol (PEG) or hyperbranched polyglycerol (HPG)). The antimicrobial activity of the 73-derived peptides was enhanced 2- to 8-fold, and all the derivatives eradicated preformed Staphylococcus aureus biofilms. Formulation of the peptides with compatible polyethylene glycol (PEG)-modified phospholipid micelles alleviated toxicity toward human cells and reduced aggregation. When evaluated in vivo, the unformulated d-enantiomers aggregated when injected under the skin of mice, but micelle encapsulated peptides were well absorbed. Pegylated micelle formulated peptides were investigated for their potential as therapeutic agents for treating high-density infections in a murine cutaneous abscess model. Formulated peptide 73 reduced abscess size by 36% and bacterial loads by 2.2-fold compared to the parent peptide aurein 2.2Δ3. Micelle encapsulated peptides 73c and D-73 exhibited superior activity, further reducing abscess sizes by 85% and 63% and lowering bacterial loads by 510- and 9-fold compared to peptide 73.

Mechanisms of Action for Antimicrobial Peptides With Antibacterial and Antibiofilm Functions.

The antibiotic crisis has led to a pressing need for alternatives such as antimicrobial peptides (AMPs). Recent work has shown that these molecules have great potential not only as antimicrobials, but also as antibiofilm agents, immune modulators, anti-cancer agents and anti-inflammatories. A better understanding of the mechanism of action (MOA) of AMPs is an important part of the discovery of more potent and less toxic AMPs. Many models and techniques have been utilized to describe the MOA. This review will examine how biological assays and biophysical methods can be utilized in the context of the specific antibacterial and antibiofilm functions of AMPs.

Filamentous Bacteriophage Proteins and Assembly.

Filamentous bacteriophages, also known as filamentous bacterial viruses or Inoviruses, have been studied extensively over the years. They are interesting paradigms in structural molecular biology and offer insight into molecular assembly, a process that remains to be fully understood. In this chapter, an overview on filamentous bacteriophages will be provided. In particular, we review the constituent proteins of filamentous bacteriophage and discuss assembly by examining the structure of the major coat protein at various stages of the process. The minor coat proteins will also be briefly reviewed. Structural information provides key snapshots into the dynamic process of assembly.

Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo.

Antibiotic resistance is projected as one of the greatest threats to human health in the future and hence alternatives are being explored to combat resistance. Antimicrobial peptides (AMPs) have shown great promise, because use of AMPs leads bacteria to develop no or low resistance. In this review, we discuss the diversity, history and the various mechanisms of action of AMPs. Although many AMPs have reached clinical trials, to date not many have been approved by the US Food and Drug Administration (FDA) due to issues with toxicity, protease cleavage and short half-life. Some of the recent strategies developed to improve the activity and biocompatibility of AMPs, such as chemical modifications and the use of delivery systems, are also reviewed in this article.