Direct and Ultrasensitive Bioluminescent Detection of Intact Respiratory Viruses.

Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of the intact virus. Instead, specific detection of intact respiratory virus particles may be more effective at assessing the contagiousness of a patient.

Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system.

The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms.

Chemical Stability of mRNA/cDNA Complexes: Defining the Limits of mRNA Display.

Messenger RNA (mRNA) display is being increasingly adopted for peptide drug candidate discovery. While many conditions have been reported for the affinity enrichment step and in some cases for peptide modification, there is still limited understanding about the versatility of peptide-puromycin-mRNA/cDNA (complementary DNA) complexes. This work explores the chemical stability of mRNA/cDNA hybrid complexes under a range of different fundamental chemical conditions as well as with peptide modification conditions reported in an mRNA display setting.

Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis.

Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge.

Synthetic Heparanase Inhibitors Can Prevent Herpes Simplex Viral Spread.

Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1.

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein.

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics.

An Efficient, Site-Selective and Spontaneous Peptide Macrocyclisation During in vitro Translation.

Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work, a new approach to peptide macrocyclisation is reported, using a cyanobenzothiazole-containing amino acid that can be incorporated into peptides by both in vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by-products, is selective over other amino acids including non N-terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions.

Inhibition of H1 and H5 Influenza A Virus Entry by Diverse Macrocyclic Peptides Targeting the Hemagglutinin Stem Region.

Influenza A viruses pose a serious pandemic risk, while generation of efficient vaccines against seasonal variants remains challenging. There is thus a pressing need for new treatment options. We report here a set of macrocyclic peptides that inhibit influenza A virus infection at low nanomolar concentrations by binding to hemagglutinin, selected using ultrahigh-throughput screening of a diverse peptide library.

Phosphine addition to dehydroalanine for peptide modification.

Thiols are a functional group commonly used for selective reactions in a biochemical setting because of their high nucleophilicity. Phosphorus nucleophiles can undergo some similar reactions to thiols, but remain underexploited in this setting. In this work we show that phosphine nucleophiles react cleanly and quickly with a dehydroalanine electrophile, itself generated from cysteine, to give a stable adduct in a peptide context.

Opportunities for Expanding Encoded Chemical Diversification and Improving Hit Enrichment in mRNA-Displayed Peptide Libraries.

DNA-encoded small-molecule libraries and mRNA displayed peptide libraries both use numerically large pools of oligonucleotide-tagged molecules to identify potential hits for protein targets. They differ dramatically, however, in the 'drug-likeness' of the molecules that each can be used to discover. We give here an overview of the two techniques, comparing some advantages and disadvantages of each, and suggest areas where particularly mRNA display can benefit from adopting advances developed with DNA-encoded small molecule libraries.

Crystal structure of the Propionibacterium acnes surface sialidase, a drug target for P. acnes-associated diseases.

Propionibacterium acnes, though generally considered part of the normal flora of human skin, is an opportunistic pathogen associated with acne vulgaris as well as other diseases, including endocarditis, endophthalmitis and prosthetic joint infections. Its virulence potential is also supported by knowledge gained from its sequenced genome. Indeed, a vaccine targeting a putative cell wall-anchored P.

Development of a 1,2-difluorofucoside activity-based probe for profiling GH29 fucosidases.

GH29 α-l-fucosidases catalyze hydrolysis of terminal α-l-fucosyl linkages with varying specificity and are expressed by prominent members of the human gut microbiota. Both homeostasis and dysbiosis at the human intestinal microbiota interface have been correlated with altered fucosidase activity. Herein we describe the development of a 2-deoxy-2-fluoro fucosyl fluoride derivative with an azide mini-tag as an activity-based probe (ABP) for selective in vitro labelling of GH29 α-l-fucosidases.

Mimicking the Nucleosomal Context in Peptide-Based Binders of a H3K36me Reader Increases Binding Affinity While Altering the Binding Mode.

Targeting of proteins in the histone modification machinery has emerged as a promising new direction to fight disease. The search for compounds that inhibit proteins that readout histone modification has led to several new epigenetic drugs, mostly for proteins involved in recognition of acetylated lysines. However, this approach proved to be a challenging task for methyllysine readers, which typically feature shallow binding pockets.

C-Terminal Tag Location Hampers in Vitro Profiling of OGT Peptide Substrates by mRNA Display.

O-GlcNAc transferase (OGT) is the only enzyme that catalyzes the post-translational modification of proteins at Ser/Thr with a single β-N-acetylglucosamine (O-GlcNAcylation). Its activity has been associated with chronic diseases such as cancer, diabetes and neurodegenerative disease. Although numerous OGT substrates have been identified, its accepted substrate scope can still be refined.

Suppression of Formylation Provides an Alternative Approach to Vacant Codon Creation in Bacterial In Vitro Translation.

Genetic code reprogramming is a powerful approach to controlled protein modification. A remaining challenge, however, is the generation of vacant codons. We targeted the initiation machinery of E.

Discovery of Potent Cyclic Sulfopeptide Chemokine Inhibitors via Reprogrammed Genetic Code mRNA Display.

Targeting chemokine signaling is an attractive avenue for the treatment of inflammatory disorders. Tyrosine sulfation is an important post-translational modification (PTM) that enhances chemokine-receptor binding and is also utilized by a number of pathogenic organisms to improve the binding affinity of immune-suppressive chemokine binding proteins (CKBPs). Here we report the display selection of tyrosine-sulfated cyclic peptides using a reprogrammed genetic code to discover high-affinity ligands for the chemokine CCL11 (eotaxin-1).

Folding Then Binding vs Folding Through Binding in Macrocyclic Peptide Inhibitors of Human Pancreatic α-Amylase.

macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and β-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized.

High-Throughput Approaches in Carbohydrate-Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery.

Carbohydrates are attached and removed in living systems through the action of carbohydrate-active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse.

Towards Tuneable Retaining Glycosidase-Inhibiting Peptides by Mimicry of a Plant Flavonol Warhead.

Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in-depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α-amylase (HPA).

Rapid Discovery of Potent and Selective Glycosidase-Inhibiting De Novo Peptides.

Human pancreatic α-amylase (HPA) is responsible for degrading starch to malto-oligosaccharides, thence to glucose, and is therefore an attractive therapeutic target for the treatment of diabetes and obesity. Here we report the discovery of a unique lariat nonapeptide, by means of the RaPID (Random non-standard Peptides Integrated Discovery) system, composed of five amino acids in a head-to-side-chain thioether macrocycle and a further four amino acids in a 3 helical C terminus. This is a potent inhibitor of HPA (K = 7 nM) yet exhibits selectivity for the target over other glycosidases tested.

Mechanistic investigations of unsaturated glucuronyl hydrolase from Clostridium perfringens.

Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct (1)H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme.

Mechanistic insights from substrate preference in unsaturated glucuronyl hydrolase.

Natural and synthetic unsaturated glucuronides were tested as substrates for Clostridium perfringens unsaturated glucuronyl hydrolase to probe its mechanism and to guide inhibitor design. Of the natural substrates, a chondroitin disaccharide substrate with sulfation of the primary alcohol on carbon 6 of its N-acetylgalactosamine moiety was found to have the highest turnover number of any substrate reported for an unsaturated glucuronyl hydrolase, with kcat =112 s(-1) . Synthetic aryl glycoside substrates with electron-withdrawing aglycone substituents were cleaved more slowly than those with electron-donating substituents.

Unusual enzymatic glycoside cleavage mechanisms.

Over the sixty years since Koshland initially formulated the classical mechanisms for retaining and inverting glycosidases, researchers have assembled a large body of supporting evidence and have documented variations of these mechanisms. Recently, however, researchers have uncovered a number of completely distinct mechanisms for enzymatic cleavage of glycosides involving elimination and/or hydration steps. In family GH4 and GH109 glycosidases, the reaction proceeds via transient NAD(+)-mediated oxidation at C3, thereby acidifying the proton at C2 and allowing for elimination across the C1-C2 bond.

The 'mirror-image' postulate as a guide to the selection and evaluation of pyrrolidines as α-l-fucosidase inhibitors.

The ability of a series of pyrrolidines to inhibit several glycosidases was investigated. Using Fleet's 'mirror-image postulate', it was proposed that enantiomeric derivatives of 1,4-dideoxy-1,4-imino-d-lyxitol (a known α-d-galactosidase inhibitor) would show inhibitory activity against α-l-fucosidases. Some modest α-l-fucosidase inhibitory activity was observed for selected compounds (particularly an aminomethyl pyrrolidine) and it was proposed that better activity could be obtained by modifying the C-2 side chain of the pyrrolidine core.