Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses

Antimicrobial and Cell-Penetrating Peptides: Understanding Penetration for the Design of Novel Conjugate Antibiotics.

Antimicrobial peptides (AMPs) are short oligopeptides that can penetrate the bacterial inner and outer membranes. Together with cell-penetrating peptides (CPPs), they are called membrane active peptides; peptides which can translocate across biological membranes. Over the last fifty years, attempts have been made to understand the molecular features that drive the interactions of membranes with membrane active peptides. This review examines the features of a membrane these peptides exploit for translocation, as well as the physicochemical characteristics of membrane active peptides which are important for translocation. Moreover, it presents examples of how these features have been used in recent years to create conjugates consisting of a membrane active peptide, called a "vector", attached to either a current or novel antibiotic, called a "cargo" or "payload". In addition, the review discusses what properties may contribute to an ideal peptide vector able to deliver cargoes across the bacterial outer membrane as the rising issue of antimicrobial resistance demands new strategies to be employed to combat this global public health threat

DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles

Integral membrane proteins (IMPs) are central to many physiological processes and represent ∼60% of current drug targets. An intricate interplay with the lipid molecules in the cell membrane is known to influence the stability, structure and function of IMPs. Detergents are commonly used to solubilize and extract IMPs from cell membranes. However, due to the loss of the lipid environment, IMPs usually tend to be unstable and lose function in the continuous presence of detergent. To overcome this problem, various technologies have been developed, including protein engineering by mutagenesis to improve IMP stability, as well as methods to reconstitute IMPs into detergent-free entities, such as nanodiscs based on apolipoprotein A or its membrane scaffold protein (MSP) derivatives, amphipols, and styrene-maleic acid copolymer-lipid particles (SMALPs). Although significant progress has been made in this field, working with inherently unstable human IMP targets (e.g., GPCRs, ion channels and transporters) remains a challenging task. Here, we present a novel methodology, termed DirectMX (for direct membrane extraction), taking advantage of the saposin-lipoprotein (Salipro) nanoparticle technology to reconstitute fragile IMPs directly from human crude cell membranes. We demonstrate the applicability of the DirectMX methodology by the reconstitution of a human solute carrier transporter and a wild-type GPCR belonging to the human chemokine receptor (CKR) family. We envision that DirectMX bears the potential to enable studies of IMPs that so far remained inaccessible to other solubilization, stabilization or reconstitution methods.QC 20211008</p

Conditional ablation of neurones in transgenic mice

Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo. This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation. Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo. As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter. We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost. The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic. Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by “knock-in” gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo

A novel stereocilia defect in sensory hair cells of the deaf mouse mutant Tasmanian devil

Stereocilia are specialized actin-filled, finger-like processes arrayed in rows of graded heights to form a crescent or W-shape on the apical surface of sensory hair cells. The stereocilia are deflected by the vibration of sound, which opens transduction channels and allows an influx of ions to depolarize the hair cell, in turn triggering synaptic activity. The specialized morphology and organization of the stereocilia bundle is crucial in the process of sensory transduction in the inner ear. However, we know little about the development of stereocilia in the mouse and few molecules that are involved in stereocilia maturation are known. We describe here a new mouse mutant with abnormal stereocilia development. The Tasmanian devil (tde) mouse mutation arose by insertional mutagenesis and has been mapped to the middle of chromosome 5. Homozygotes show head-tossing and circling and have raised thresholds for cochlear nerve responses to sound. The gross morphology of the inner ear was normal, but the stereocilia of cochlear and vestibular hair cells are abnormally thin, and they become progressively disorganized with increasing age. Ultimately, the hair cells die. This is the first report of a mutant showing thin stereocilia. The association of thin stereocilia with cochlear dysfunction emphasizes the critical role of stereocilia in auditory transduction, and the discovery of the Tasmanian devil mutant provides a resource for the identification of an essential molecule in hair cell function

Structure-Guided Chemical Optimization of Bicyclic Peptide (<i>Bicycle</i>) Inhibitors of Angiotensin-Converting Enzyme 2

Angiotensin-converting enzyme 2 (ACE2) is a metalloprotease that cleaves angiotensin II, a peptide substrate involved in the regulation of hypertension. Here, we identified a series of constrained bicyclic peptides, Bicycle, inhibitors of human ACE2 by panning highly diverse bacteriophage display libraries. These were used to generate X-ray crystal structures which were used to inform the design of additional Bicycles with increased affinity and inhibition of ACE2 enzymatic activity. This novel structural class of ACE2 inhibitors is among the most potent ACE2 inhibitors yet described in vitro, representing a valuable tool to further probe ACE2 function and for potential therapeutic utility

Lipid-rich enteral nutrition regulates mucosal mast cell activation via the vagal anti-inflammatory reflex

Item does not contain fulltextNutritional stimulation of the cholecystokinin-1 receptor (CCK-1R) and nicotinic acetylcholine receptor (nAChR)-mediated vagal reflex was shown to reduce inflammation and preserve intestinal integrity. Mast cells are important early effectors of the innate immune response; therefore modulation of mucosal mast cells is a potential therapeutic target to control the acute inflammatory response in the intestine. The present study investigates intestinal mast cell responsiveness upon nutritional activation of the vagal anti-inflammatory reflex during acute inflammation. Mucosal mast cell degranulation was induced in C57/Bl6 mice by administration of Salmonella enterica LPS. Lipid-rich enteral feeding prior to LPS significantly decreased circulatory levels of mouse mast cell protease at 30 min post-LPS compared with isocaloric low-lipid nutrition or fasting. CCK-1R blockage reversed the inhibitory effects of lipid-rich feeding, whereas stimulation of the peripheral CCK-1R mimicked nutritional mast cell inhibition. The effects of lipid-rich nutrition were negated by nAChR blockers chlorisondamine and alpha-bungarotoxin and vagal intestinal denervation. Accordingly, release of beta-hexosaminidase by MC/9 mast cells following LPS or IgE-ovalbumin complexes was dose dependently inhibited by acetylcholine and nicotine. Application of GSK1345038A, a specific agonist of the nAChR alpha7, in bone marrow-derived mast cells from nAChR beta2-/- and wild types indicated that cholinergic inhibition of mast cells is mediated by the nAChR alpha7 and is independent of the nAChR beta2. Together, the present study reveals mucosal mast cells as a previously unknown target of the nutritional anti-inflammatory vagal reflex