Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Amide- and bis-amide-linked highly potent and broadly active antifungal agents for the treatment of invasive fungal infections- towards the discovery of pre-clinical development candidate FC12406.

Most fungal infections are common, localized to skin or mucosal surfaces and can be treated effectively with topical antifungal agents. However, while invasive fungal infections (IFIs) are uncommon, they are very difficult to control medically, and are associated with high mortality rates. We have previously described highly potent bis-guanidine-containing heteroaryl-linked antifungal agents, and were interested in expanding the range of agents to novel series so as to reduce the degree of aromaticity (with a view to making the compounds more drug-like), and provide broadly active high potency derivatives. We have investigated the replacement of the central aryl ring from our original series by both amide and a bis-amide moieties, and have found particular structure-activity relationships (SAR) for both series', resulting in highly active antifungal agents against both mold and yeast pathogens. In particular, we describe the in vitro antifungal activity, absorption, distribution, metabolism and elimination (ADME) properties, and off-target properties of FC12406 (34), which was selected as a pre-clinical development candidate.

Highly potent, broadly active antifungal agents for the treatment of invasive fungal infections.

Invasive fungal infections have become an important healthcare issue due in large part to high mortality rates under standard of care (SOC) therapies creating an urgent need for new and effective anti-fungal agents. We have developed a series of non-peptide, structurally-constrained analogs of host defence proteins that have distinct advantages over peptides for pharmaceutical uses. Here we report the chemical optimization of bis-guanidine analogs focused on alterations of the central aryl core and the connection of it to the terminal guanidines. This effort resulted in the production of highly potent, broadly active compounds with low mammalian cell cytotoxicity that have comparable or improved antifungal activities over SOC agents. One optimal compound was also found to possess favourable in vitro pharmaceutical and off-target properties suitable for further development.

Acinetobacter baumannii OxPhos inhibitors as selective anti-infective agents.

The Gram-negative bacterium Acinetobacter baumannii is an opportunistic pathogen in humans and infections are poorly treated by current therapy. Recent emergence of multi-drug resistant strains and the lack of new antibiotics demand an immediate action for development of new anti-Acinetobacter agents. To this end, oxidative phosphorylation (OxPhos) was identified as a novel target for drug discovery research. Consequently, a library of ∼10,000 compounds was screened using a membrane-based ATP synthesis assay. One hit identified was the 2-iminobenzimidazole 1 that inhibited the OxPhos of A. baumannii with a modestly high selectivity against mitochondrial OxPhos, and displayed an MIC of 25µM (17µg/mL) against the pathogen. The 2-iminobenzimidazole 1 was found to inhibit the type 1 NADH-quinone oxidoreductase (NDH-1) of A. baumannii OxPhos by a biochemical approach. Among various derivatives that were synthesized to date, des-hydroxy analog 5 is among the most active with a relatively tight SAR requirement for the N'-aminoalkyl side chain. Analog 5 also showed less cytotoxicity against NIH3T3 and HepG2 mammalian cell lines, demonstrating the potential for this series of compounds as anti-Acinetobacter agents. Additional SAR development and target validation is underway.

Activity of potent and selective host defense peptide mimetics in mouse models of oral candidiasis.

There is a strong need for new broadly active antifungal agents for the treatment of oral candidiasis that not only are active against many species of Candida, including drug-resistant strains, but also evade microbial countermeasures which may lead to resistance. Host defense peptides (HDPs) can provide a foundation for the development of such agents. Toward this end, we have developed fully synthetic, small-molecule, nonpeptide mimetics of the HDPs that improve safety and other pharmaceutical properties. Here we describe the identification of several HDP mimetics that are broadly active against C. albicans and other species of Candida, rapidly fungicidal, and active against yeast and hyphal cultures and that exhibit low cytotoxicity for mammalian cells. Importantly, specificity for Candida over commensal bacteria was also evident, thereby minimizing potential damage to the endogenous microbiome which otherwise could favor fungal overgrowth. Three compounds were tested as topical agents in two different mouse models of oral candidiasis and were found to be highly active. Following single-dose administrations, total Candida burdens in tongues of infected animals were reduced up to three logs. These studies highlight the potential of HDP mimetics as a new tool in the antifungal arsenal for the treatment of oral candidiasis.

Potency of a small molecule that targets the molluscum contagiosum virus processivity factor increases when conjugated to a tripeptide.

We recently developed compound FC-7269 for targeting the Molluscum contagiosum virus processivity factor (mD4) and demonstrated its ability to inhibit viral processive DNA synthesis in vitro and cellular infection of an mD4-dependent virus (Antiviral Res 211, 2023,105520). However, despite a thorough medicinal chemistry campaign we were unable to generate a potent second analog as a requisite for drug development. We overcame this impasse, by conjugating a short hydrophobic trivaline peptide to FC-7269 to produce FC-TriVal-7269 which significantly increased antiviral potency and reduced cellular toxicity.

A small molecule that targets the processivity factor of molluscum contagiosum virus has therapeutic potential.

Molluscum contagiosum (MC) is an infectious disease that occurs only in humans with a tropism that is narrowly restricted to the outermost epidermal layer of the skin. Molluscum contagiosum virus (MCV) is the causative agent of MC which produces skin lesions that can persist for months to several years. MCV is efficiently transmitted by direct physical contact or by indirect contact with fomites. MC is most prevalent in children and immune compromised patients. The failure to develop a drug that targets MCV replication has been hampered for decades by the inability to propagate MCV in cell culture. To address this dilemma, we recently engineered a surrogate poxvirus expressing the MCV processivity factor (mD4) as the drug target. The mD4 protein is essential for viral replication by keeping the viral polymerase tethered to the DNA template. In this study we have designed and synthesized a lead compound (7269) that is able to prevent mD4 dependent processive DNA synthesis in vitro (IC50 = 6.8 µM) and effectively inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells (EC50 = 13.2 µM) with negligible cytotoxicity. In human liver microsomes, 7269 was shown to be stable for almost 2 h. When tested for penetration into human cadaver skin in a formulated gel, the level of 7269 in the epidermal layer was nearly 100 times the concentration (EC50) needed to inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells. The gel formulated 7269 was scored as a non-irritant on skin and shown to have a shelf-life that was completely stable after several months. In summary, 7269 is a potential Lead for becoming the first MCV anti-viral compound to treat MC and thereby, addresses this unmet medical need that has persisted for many decades.

Synthetic mimics of antimicrobial peptides with immunomodulatory responses.

A new series of aryl-based synthetic mimics of antimicrobial peptides (SMAMPs) with antimicrobial activity and selectivity have been developed via systematic tuning of the aromatic groups and charge. The addition of a pendant aromatic group improved the antimicrobial activity against Gram-negative bacteria, while the addition of charge improved the selectivity. SMAMP 4 with six charges and a naphthalene central ring demonstrated a selectivity of 200 against both Staphylococcus aureus and Escherichia coli , compared with a selectivity of 8 for the peptide MSI-78. In addition to the direct antimicrobial activity, SMAMP 4 exhibited specific immunomodulatory activities in macrophages both in the presence and in the absence of lipopolysaccharide, a TLR agonist. SMAMP 4 also induced the production of a neutrophil chemoattractant, murine KC, in mouse primary cells. This is the first nonpeptidic SMAMP demonstrating both good antimicrobial and immunomodulatory activities.

New bactericidal surgical suture coating.

This paper demonstrates the effectiveness of a new antimicrobial suture coating. An amphiphilic polymer, poly[(aminoethyl methacrylate)-co-(butyl methacrylate)] (PAMBM), inspired by antimicrobial peptides, was bactericidal against S. aureus in time-kill experiments. PAMBM was then evaluated in a variety of polymer blends using the Japanese Industrial Standard (JIS) method and showed excellent antimicrobial activity at a low concentration (0.5 wt %). Using a similar antimicrobial coating formula to commercial Vicryl Plus sutures, disk samples of the coating material containing PAMBM effectively killed bacteria (98% reduction at 0.75 wt %). Triclosan, the active ingredient in Vicryl Plus coatings, did not kill the bacteria. Further Kirby-Bauer assays of these disk samples showed an increasing zone of inhibition with increasing concentration of PAMBM. Finally, the PAMBM-containing coating was applied to sutures, and the morphology of the coating surface was characterized by SEM, along with Vicryl and uncoated sutures. The PAMBM-containing sutures killed bacteria more effectively (3 log(10) reduction at 2.4 wt %) than Vicryl Plus sutures (0.5 log(10) reduction).

Anion mediated activation of guanidine rich small molecules.

Cell penetrating peptides (CPPs) and their synthetic analogs are of widespread interest. Here we report that guanidine rich small molecules can be potential membrane transporters in the presence of hydrophobic counteranion activators. To our knowledge, this is the first example of small molecules that mimic the anion-activated transport function of CPP.

De novo design and in vivo activity of conformationally restrained antimicrobial arylamide foldamers.

The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics.

De novo design of antimicrobial polymers, foldamers, and small molecules: from discovery to practical applications.

Antimicrobial peptides (AMPs) provide protection against a variety of pathogenic bacteria and are, therefore, an important part of the innate immune system. Over the past decade, there has been considerable interest in developing AMPs as intravenously administered antibiotics. However, despite extensive efforts in the pharmaceutical and biotechnology industry, it has proven difficult to achieve this goal. While researchers have solved some relatively simple problems such as susceptibility to proteolysis, more severe problems have included the expense of the materials, toxicity, poor efficacy, and limited tissue distribution. In this Account, we describe our efforts to design and synthesize "foldamers"-- short sequence-specific oligomers based on arylamide and beta-amino acid backbones, which fold into well-defined secondary structures-- that could act as antimicrobial agents. We reasoned that small "foldamers" would be less expensive to produce than peptides, and might have better tissue distribution. It should be easier to fine-tune the structures and activities of these molecules to minimize toxicity. Because the activities of many AMPs depends primarily on their overall physicochemical properties rather than the fine details of their precise amino acid sequences, we have designed and synthesized very small "coarse-grained" molecules, which are far simpler than naturally produced AMPs. The molecular design of these foldamers epitomizes the positively charged amphiphilic structures believed to be responsible for the activity of AMPs. The designed oligomers show greater activity than the parent peptides. They have also provided leads for novel small molecule therapeutics that show excellent potency in animal models for multidrug resistant bacterial infections. In addition, such molecules can serve as relatively simple experimental systems for investigations aimed at understanding the mechanism of action for this class of antimicrobial agents. The foldamers' specificity for bacterial membranes relative to mammalian membranes appears to arise from differences in membrane composition and physical properties between these cell types. Furthermore, because experimental coarse-graining provided such outstanding results, we developed computational coarse-grained models to enable molecular dynamic simulations of these molecules with phospholipid membranes. These simulations allow investigation of larger systems for longer times than conventional molecular dynamics simulations, allowing us to investigate how physiologically relevant surface concentrations of AMP mimics affect the bilayer structure and properties. Finally, we apply the principles discovered through this work to the design of inexpensive antimicrobial polymers and materials.

Changes in Leaf-Level Nitrogen Partitioning and Mesophyll Conductance Deliver Increased Photosynthesis for Lolium perenne Leaves Engineered to Accumulate Lipid Carbon Sinks.

Diacylglycerol acyl-transferase (DGAT) and cysteine oleosin (CO) expression confers a novel carbon sink (of encapsulated lipid droplets) in leaves of Lolium perenne and has been shown to increase photosynthesis and biomass. However, the physiological mechanism by which DGAT + CO increases photosynthesis remains unresolved. To evaluate the relationship between sink strength and photosynthesis, we examined fatty acids (FA), water-soluble carbohydrates (WSC), gas exchange parameters and leaf nitrogen for multiple DGAT + CO lines varying in transgene accumulation. To identify the physiological traits which deliver increased photosynthesis, we assessed two important determinants of photosynthetic efficiency, CO2 conductance from atmosphere to chloroplast, and nitrogen partitioning between different photosynthetic and non-photosynthetic pools. We found that DGAT + CO accumulation increased FA at the expense of WSC in leaves of L. perenne and for those lines with a significant reduction in WSC, we also observed an increase in photosynthesis and photosynthetic nitrogen use efficiency. DGAT + CO L. perenne displayed no change in rubisco content or Vcmax but did exhibit a significant increase in specific leaf area (SLA), stomatal and mesophyll conductance, and leaf nitrogen allocated to photosynthetic electron transport. Collectively, we showed that increased carbon demand via DGAT+CO lipid sink accumulation can induce leaf-level changes in L. perenne which deliver increased rates of photosynthesis and growth. Carbon sinks engineered within photosynthetic cells provide a promising new strategy for increasing photosynthesis and crop productivity.

Herpes Simplex Virus-1 infection in human primary corneal epithelial cells is blocked by a stapled peptide that targets processive DNA synthesis.

PURPOSE: Acyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus -1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. METHODS: A series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. RESULTS: The stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. CONCLUSIONS: Hydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.

Elevation of oil body integrity and emulsion stability by polyoleosins, multiple oleosin units joined in tandem head-to-tail fusions.

We have successfully created polyoleosins by joining multiple oleosin units in tandem head-to-tail fusions. Constructs encoding recombinant proteins of 1, 3 and 6 oleosin repeats were purposely expressed both in planta and in Escherichia coli. Recombinant polyoleosins accumulated in the seed oil bodies of transgenic plants and in the inclusion bodies of E. coli. Although polyoleosin was estimated to only accumulate to <2% of the total oil body protein in planta, their presence increased the freezing tolerance of imbibed seeds as well as emulsion stability and structural integrity of purified oil bodies; these increases were greater with increasing oleosin repeat number. Interestingly, the hexameric form of polyoleosin also led to an observable delay in germination which could be overcome with the addition of external sucrose. Prokaryotically produced polyoleosin was purified and used to generate artificial oil bodies and the increase in structural integrity of artificial oil bodies-containing polyoleosin was found to mimic those produced in planta. We describe here the construction of polyoleosins, their purification from E. coli, and properties imparted on seeds as well as native and artificial oil bodies. A putative mechanism to account for these properties is also proposed.

A Novel Immunocompetent Mouse Model for Testing Antifungal Drugs Against Invasive Candida albicans Infection.

Disseminated infection by Candida species represents a common, often life-threatening condition. Increased resistance to current antifungal drugs has led to an urgent need to develop new antifungal drugs to treat this pathogen. However, in vivo screening of candidate antifungal compounds requires large numbers of animals and using immunosuppressive agents to allow for fungal dissemination. To increase the efficiency of screening, to use fewer mice, and to remove the need for immunosuppressive agents, which may interfere with the drug candidates, we tested the potential for a novel approach using in vivo imaging of a fluorescent strain of Candida albicans, in a mouse strain deficient in the host defense peptide, murine ß-defensin 1 (mBD-1). We developed a strain of C. albicans that expresses red fluorescent protein (RFP), which exhibits similar infectivity to the non-fluorescent parent strain. When this strain was injected into immunocompetent mBD-1-deficient mice, we observed a non-lethal disseminated infection. Further, we could quantify its dissemination in real time, and observe the activity of an antifungal peptide mimetic drug by in vivo imaging. This novel method will allow for the rapid in vivo screening of antifungal drugs, using fewer mice, and increase the efficiency of testing new antifungal agents.

The Genome Sequence of the Anthelmintic-Susceptible New Zealand Haemonchus contortus.

Internal parasitic nematodes are a global animal health issue causing drastic losses in livestock. Here, we report a H. contortus representative draft genome to serve as a genetic resource to the scientific community and support future experimental research of molecular mechanisms in related parasites. A de novo hybrid assembly was generated from PCR-free whole genome sequence data, resulting in a chromosome-level assembly that is 465 Mb in size encoding 22,341 genes. The genome sequence presented here is consistent with the genome architecture of the existing Haemonchus species and is a valuable resource for future studies regarding population genetic structures of parasitic nematodes. Additionally, comparative pan-genomics with other species of economically important parasitic nematodes have revealed highly open genomes and strong collinearities within the phylum Nematoda.

Small-Molecule Host-Defense Peptide Mimetic Antibacterial and Antifungal Agents Activate Human and Mouse Mast Cells via Mas-Related GPCRs.

Host-defense peptides (HDPs) have an important therapeutic potential against microbial infections but their metabolic instability and cellular cytotoxicity have limited their utility. To overcome these limitations, we utilized five small-molecule, nonpeptide HDP mimetics (smHDPMs) and tested their effects on cytotoxicity, antimicrobial activity, and mast cell (MC) degranulation. None of the smHDPMs displayed cytotoxicity against mouse 3T3 fibroblasts or human transformed liver HepG2 cells. However, one compound had both antifungal and antibacterial activity. Surprisingly, all five compounds induced degranulation in a human MC line, LAD2, and this response was substantially reduced in Mas-related G protein-coupled receptor (GPCR)-X2 (MRGPRX2)-silenced cells. Furthermore, all five compounds induced degranulation in RBL-2H3 cells expressing MRGPRX2 but this response was abolished in cells expressing naturally occurring loss-of-function missense variants G165E (rs141744602) and D184H (rs372988289). Mrgprb2 is the likely mouse ortholog of human MRGPRX2, which is expressed in connective tissue MCs (CTMCs) such as cutaneous and peritoneal MCs (PMCs). All five smHDPMs induced degranulation in wild-type PMCs but not in cells derived from Mrgprb2⁻/⁻ mice. These findings suggest that smHDPMs could serve as novel targets for the treatment of drug-resistant fungal and bacterial infections because of their ability to harness CTMCs' host defense functions.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor.

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox. The VACV processivity factor D4 is an ideal therapeutic target since it is both essential and specific for poxvirus replication. Recently, we identified a tripeptide (Gly-Phe-Ile) motif at the C-terminus of D4 that is conserved among poxviruses and is necessary for maintaining protein function. In the current work, a virtual screening for small molecule mimics of the tripeptide identified a thiophene lead that effectively inhibited VACV, cowpox virus, and rabbitpox virus in cell culture (EC50 = 8.4-19.7 µM) and blocked in vitro processive DNA synthesis (IC50 = 13.4 µM). Compound-binding to D4 was demonstrated through various biophysical methods and a dose-dependent retardation of the proteolysis of D4 proteins. This study highlights an inhibitor design strategy that exploits a susceptible region of the protein and identifies a novel scaffold for a broad-spectrum poxvirus inhibitor.