Expanding the toolbox for the study of antimicrobial peptides

There is an urgent lack of new antibiotics in the face of an ever-expanding antimicrobial resistance crisis. The fact that fewer new classes of antibiotics are being developed, and resistance soon follows newly available antibiotics, only serves to underline the urgency of the matter. There is a clear need of a paradigm shift with regards to antibiotics, and one such hope is antimicrobial peptides (AMPs). AMPs are an integral part of the innate immune systems of most organisms within the domains of life; since their discovery they have become of significant interest as a new type of antimicrobial agent, due in part to the low capacity of bacteria to develop resistance mechanisms towards them. Despite their potential, and lengthy study so far, establishing the specifics of the mechanism of action of many AMPs remains difficult– particularly of those that target the bacterial cell membrane. This lack of understanding limits the ability to rationally design new AMPs with a view to developing new antimicrobial agents. The aim of this work was to help identify new potential hit compounds through NMR structure elucidation, and to develop new methods that would give greater insight into the activity of membrane active AMPs. This in turn could help enable the rational design of new AMPs. WIND-PVPA, a method to quantify permeabilities of water and ions as a means to evaluate the disruptive capabilities of AMPs, was developed. This was demonstrated on a number of AMPs, and it was shown that WIND-PVPA can identify AMPs that have strong, selective, membrane disruptive activities such as the AMP WRWRWR, as well as more modestly disruptive AMPs such as KP-76. The WIND-PVPA was further used with a non-AMP membrane active natural product – lulworthinone – that was characterised over the course of the project. The findings of the study helped classify lulworthinone as a non-disruptive membrane active agent. In addition, microscale thermophoresis (MST) was shown to be a viable method by which the binding and partition coefficients of Trp-rich AMPs can be determined, and it was shown that the derived lipid-bindings of the AMPs correlates well with their bactericidal activity. Both WIND-PVPA and MST have expanded the toolbox available to the study of AMP-lipid interactions and can be used synergistically to give greater insight into the possible mechanism by which AMPs act, by helping to identify interesting cases, such as non-disruptive AMPs with potent activities. In summary, the methods developed have great potential that can be further refined into robust methods that can greatly assist in the deconvolution of AMP activity and can open up possibilities of the rational design of membrane active AMPs as a new generation of antimicrobial agents.

Lulworthinone, a New Dimeric Naphthopyrone From a Marine Fungus in the Family Lulworthiaceae With Antibacterial Activity Against Clinical Methicillin-Resistant Staphylococcus aureus Isolates

The emergence of drug-resistant bacteria is increasing rapidly in all parts of the world, and the need for new antibiotics is urgent. In our continuous search for new antimicrobial molecules from under-investigated Arctic marine microorganisms, a marine fungus belonging to the family Lulworthiaceae (Lulworthiales, Sordariomycetes, and Ascomycota) was studied. The fungus was isolated from driftwood, cultivated in liquid medium, and studied for its potential for producing antibacterial compounds. Through bioactivity-guided isolation, a novel sulfated biarylic naphtho-α-pyrone dimer was isolated, and its structure was elucidated by spectroscopic methods, including 1D and 2D NMR and HRMS. The compound, named lulworthinone (1), showed antibacterial activity against reference strains of Staphylococcus aureus and Streptococcus agalactiae, as well as several clinical MRSA isolates with MICs in the 1.56–6.25 μg/ml range. The compound also had antiproliferative activity against human melanoma, hepatocellular carcinoma, and non-malignant lung fibroblast cell lines, with IC50 values of 15.5, 27, and 32 μg/ml, respectively. Inhibition of bacterial biofilm formation was observed, but no eradication of established biofilm could be detected. No antifungal activity was observed against Candida albicans. During the isolation of 1, the compound was observed to convert into a structural isomer, 2, under acidic conditions. As 1 and 2 have high structural similarity, NMR data acquired for 2 were used to aid in the structure elucidation of 1. To the best of our knowledge, lulworthinone (1) represents the first new bioactive secondary metabolite isolated from the marine fungal order Lulworthiales

WIND-PVPA: Water/Ion NMR Detected PVPA to assess lipid barrier integrity in vitro through quantification of passive water- and ion transport

Water/Ion NMR Detected – Phospholipid Vesicle Permeability Assay (WIND-PVPA), is presented as a novel, straightforward and automatable method to assess lipid barrier integrity in vitro. The apparent permeability constants of water- and ions across the PVPA barriers are determined in a one-pot experiment under the influence of membrane-active guest molecules. NMR spectroscopy is used to quantify the water directly (D2O) and the ions indirectly (complexed with EDTA) as a function of time. WIND-PVPA is demonstrated using four anti-microbial peptides, to show that membrane active molecules can be differentiated by their disruptive influence on the PVPA system. The results obtained are compared with explicit molecular dynamics simulations of lipid bilayers, AMPs, water and salt, where the motions of all individual water molecules relative to the lipid bilayer are monitored over the course of the simulations, allowing the calculation of theoretical apparent permeability constants of the corresponding single bilayer systems. Proof-of-principle is presented that WIND-PVPA can be used to evaluate the lipid barrier destabilizing effect of active guest molecules by measuring changes in passive water- and ion permeabilities upon exposure. The method is highly flexible in terms of barrier composition, choice of probes and membrane active compounds

A turbulent decade for NSAIDs: update on current concepts of classification, epidemiology, comparative efficacy, and toxicity

Non-steroidal anti-inflammatory drugs (NSAIDs) represent a diverse class of drugs and are among the most commonly used analgesics for arthritic pain worldwide, though long-term use is associated with a spectrum of adverse effects. The introduction of cyclooxygenase-2-selective NSAIDs early in the last decade offered an alternative to traditional NSAIDs with similar efficacy and improved gastrointestinal tolerability; however, emerging concerns about cardiovascular safety resulted in the withdrawal of two agents (rofecoxib and valdecoxib) in the mid-2000s and, subsequently, in an overall reduction in NSAID use. It is now understood that all NSAIDs are associated with some varying degree of gastrointestinal and cardiovascular risk. Guidelines still recommend their use, but little is known of how patients use these agents. While strategies and guidelines aimed at reducing NSAID-associated complications exist, there is a need for evidence-based algorithms combining cardiovascular and gastrointestinal factors that can be used to aid treatment decisions at an individual patient level

CARDIAC ANOMALIES IN MONGOLISM

In a well-known textbook Maud Abbott is credited with the opinion that " the commonest cardiac anomaly in Mongolian idiocy is a defect of the interauricular septum, especially ostium primum. " Reference to her paper shows that she actually wrote that in mongols the defect " in our experience is not infrequently of this type " (Abbott, 1924). She described one case, in which there was also a ventricular septal defect that divided the aortic cusp of the mitral valve. This addition is suggestive of persistent atrioventricularis communis, of which Taussig (1947) wrote: " The diagnosis is based on the law of probability. If a Mongolian idiot has a congenital malformation of the heart which superficially resembles a ventricular septal defect and there is no cyanosis, the overwhelming probability is that he has a patent atrioventricularis communis. The demonstration of an abnormally low oxygen saturation of the arterial blood clinches the diagnosis." Robson (1931), Meecker (1935) and Robinson (1941), have described examples. Goetsch's patient showed the same lesion and dextroposition and infantile coarctation of the aorta (Goetsch, 1938), while two patients described by Gunn and Dieckmann (1927) showed mongolism, persistent atrio-ventricularis communis and patent foramen ovale, wvith patent ductus arteriosus also in one case. One might easily get the impression that persistent atrioventricularis communis was the usual cardiac lesion of mongolism, in spite of the description of the single cases with other anomalies by Wolf and Levinson (1939) and Mannheimer (1949). However, Leech (1935) described six cases, only one of which had this defect, with patent foramen ovale and anomalies of the great vessels in addition. Writers on mongolism (e.g. Benda, 1946, and Engler, 1949) do not make nuimerical statements about the frequency of the lesions they mention. The examination of a larger series than that of Leech would seem to be worth while