Synthesis of DNA-Encoded Macrocyclic Peptides via Nitrile-Aminothiol Click Reaction.

DNA-encoded library (DEL) technology holds exciting potential for discovering novel therapeutic macrocyclic peptides (MPs). Herein, we describe the development of a DEL-compatible peptide macrocyclization method that proceeds via intramolecular click-condensation between 3-(2-cyano-4-pyridyl)-l-alanine (Cpa) and an N-terminal cysteine. Cyclization takes place spontaneously in a buffered aqueous solution and affords the cyclized products in excellent yields. The reaction exhibits a broad substrate scope and can be employed to generate MPs of variable ring size and amino acid composition.

Sequential injection analysis coupled to on-line benchtop proton NMR: Method development and application to the determination of synthetic cathinones in seized drug samples.

Synthetic cathinones are a class of new psychoactive substances (NPS), an emerging group of analogues to traditional illicit drugs which are functionalized to circumvent legal regulations. The analytical investigation of NPS by traditional methods, such as gas chromatography-mass spectrometry (GC-MS), is challenging because newly emerging NPS may not yet appear in spectral libraries and because of the inability to determine certain positional isomers. Low-field or "benchtop" proton nuclear magnetic resonance spectroscopy (NMR) is an alternative that provides significant qualitative information but is particularly susceptible to matrix interferences. To this end, the development of a Sequential Injection Analysis (SIA) method which uses solid-phase extraction (SPE) to remove interfering matrix components prior to NMR determination is described. Factors including the type of SPE sorbent, column dimensions, and sample loading and elution conditions were examined. Several cathinone simulants (primary, secondary, and tertiary amines), "DEA exempt" cathinone standards, as well as authentic case samples were studied. The selectivity of the SIA-NMR-UV method was investigated against a broad range of "cutting agents" and was found to successfully remove all compounds tested with the exception of other basic drugs (e.g., acetaminophen). The limit of detection and reproducibility of the method were optimized using a Plackett-Burman screening design and Sequential Simplex optimization. Using a UV detector for dual (in series) quantification, the multivariate-optimized method produced a method limit of detection (3σ) for the cathinone simulant Phenylpropanolamine (PPA) of 23 µmol L-1, and a calibration model, in terms of UV peak area, of Area = 0.19 [PPA, mmol L-1] - 0.04. The optimized method generated ~2 mL of waste per day, and had a footprint of ~1 m2 Finally, the multivariate-optimized SIA-NMR-UV method was successfully applied to several more case samples and the cathinones were definitively identified.

NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

Oxadiazole-Containing Macrocyclic Peptides Potentiate Azole Activity against Pathogenic Candida Species.

Opportunistic pathogens of the genus Candida reign as the leading cause of mycotic disease and are associated with mortality rates greater than 40%, even with antifungal intervention. This is in part due to the limited arsenal of antifungals available to treat systemic fungal infections. Azoles have been the most widely deployed class of antifungal drug for decades and function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. However, their utility is compromised by their fungistatic nature, which favors the development of resistance. Combination therapy has the potential to confer enhanced efficacy as well as mitigate the evolution of resistance. Previously, we described the generation of structurally diverse macrocyclic peptides with a 1,3,4-oxadiazole and an endocyclic amine grafted within the peptide backbone. Importantly, this noncanonical backbone displayed high membrane permeability, an important attribute for compounds that need to permeate across the fungal cell wall and membrane in order to reach their intracellular target. Here, we explored the bioactivity of this novel chemical scaffold on its own and in combination with the azole fluconazole. Although few of the oxadiazole-containing macrocyclic peptides displayed activity against Candida albicans on their own, many increased the efficacy of fluconazole, resulting in a synergistic combination that was independent of efflux inhibition. Interestingly, these molecules also enhanced azole activity against several non-albicans Candida species, including the azole-resistant pathogens Candida glabrata and Candida auris This work characterizes a novel chemical scaffold that possesses azole-potentiating activity against clinically important Candida species.IMPORTANCE Fungal infections, such as those caused by pathogenic Candida species, pose a serious threat to human health. Treating these infections relies heavily on the use of azole antifungals; however, resistance to these drugs develops readily, demanding novel therapeutic strategies. This study characterized the antifungal activity of a series of molecules that possess unique chemical attributes and the ability to traverse cellular membranes. We observed that many of the compounds increased the activity of the azole fluconazole against Candida albicans, without blocking the action of drug efflux pumps. These molecules also increased the efficacy of azoles against other Candida species, including the emerging azole-resistant pathogen Candida auris Thus, we describe a novel chemical scaffold with broad-spectrum bioactivity against clinically important fungal pathogens.

In Vitro Dissolution Model Can Predict the in Vivo Taste Masking Performance of Coated Multiparticulates.

The majority of active pharmaceutical ingredients (APIs) are bitter. Therefore, compliance can be a problem where adequate taste masking has not been achieved; this is most problematic in pediatrics. Taste masking is thus a key stage during pharmaceutical development with an array of strategies available to the formulation scientist. Solid oral dosage forms can be taste-masked quite simply by polymer coating, which prevents drug release in the mouth, without unwantedly impairing drug release further down the gastrointestinal tract. At the early stages of pharmaceutical development, an in vitro method for the assessment of taste masking is necessary given the lack of toxicological data preventing the use of human taste panels. Currently, there is no such tool allowing prediction of taste masking efficiency. In this study, drug dissolution in the context of aversive taste thresholds was proposed as a means to bridge this knowledge gap. Thus, a biorelevant buccal dissolution test was developed in which previously determined taste thresholds in vivo were used to evaluate taste masking efficiency: if drug release exceeded said thresholds, the formulation was deemed to be poorly taste-masked, and vice versa. This novel dissolution test was compared to the USP I (basket) dissolution test, and the biopharmaceutical implications of taste masking were also assessed by performing USP I (basket) dissolution testing in simulated gastric fluid (SGF). Chlorphenamine maleate, a model bitter BCS class 1 API, was layered onto sugar spheres and taste-masked using polymer coatings. An array of coating technologies were employed and assessed single blinded: two pH-independent water-insoluble coatings (Surelease:Opadry at 8, 12, and 16% weight gain and Opadry EC at 4, 6, and 8% weight gain) and a pH-dependent water-insoluble reverse-enteric coating (developmental fully formulated system based on Kollicoat Smartseal 100P at 10% weight gain). Both the biorelevant buccal and the USP I dissolution tests were capable of discriminating between both type and level of coating used. However, only the buccal dissolution test was able to provide absolute quantification of the level of taste masking achieved in the context of previously determined taste thresholds, while the USP I test merely provided a relative comparison between the different technologies assessed. When the release data from the buccal test were assessed in parallel to that in SGF, it was possible to predict in vitro optimized taste masking without compromising bioavailability. The fully formulated system based on Smartseal 100P was identified as the most effective coating and Surelease:Opadry the least effective. The developed methodology provides true insight for the formulator, enabling more informed patient-centric formulation decisions, better taste masking, and ultimately more effective medicines.

Development of Endocyclic Control Elements for Peptide Macrocycles.

Synthetic methods that provide control over macrocycle conformation represent valuable tools for the discovery of bioactive molecules. Incorporation of heterocycles into cyclic peptides may offer a way to stabilize their solution conformations. Herein, we used N-(isocyanimino)triphenylphosphorane (Pinc) to install an oxadiazole ring and an endocyclic amine into peptide macrocycles. To elucidate the conformational effect of this constellation of functionalities, we performed synthesis, variable temperature NMR analysis, and NOE-based molecular dynamics simulation of a range of macrocycles in DMSO. As part of this study, we conducted experiments to compare macrocycle conformation in aqueous and DMSO solutions. The obtained solution structures suggest that the reduced amide bond/heterocycle (RAH) motif can stabilize macrocycle conformations in both water and DMSO, which addresses an enduring challenge in molecular design. The conformational effect of the RAH was used in an effort to mimic the biologically relevant secondary structure of MAdCAM-1. This resulted in the discovery of a novel α4ß7 integrin antagonist.

EUS-guided thrombin injection for management of gastric fundal varices.

BACKGROUND AND STUDY AIMS: Cyanoacrylate glue is recommended first-line endoscopic therapy for gastric fundal varices but it is difficult to use and carries a risk of embolization. Thrombin is preferred by many in the UK, but its effectiveness can be difficult to establish at endoscopy and the rate of re-bleeding is higher. Endoscopic ultrasound (EUS) can help assess variceal blood flow and has the potential to improve both targeting and effectiveness of injection therapy. Whereas there is already some data for its use with glue, little data currently exist in relation to its use with thrombin. PATIENTS AND METHODS: We present a series of patients treated with EUS-guided thrombin injection over the last 4 years. Thrombin was injected under EUS guidance with the intention of obliterating flow within the fundal varices. Outcomes reviewed included whether haemostasis was achieved, the dose of thrombin required for endosonographic variceal obliteration, the incidence of re-bleeding, and procedural related adverse events. RESULTS: Eight patients received EUS-guided thrombin: 3 with active bleeding and 5 as elective prevention. In 2/3 (66 %) patients with active bleeding haemostasis was achieved after a single dose with complete variceal obliteration. 1/3 (33 %) had no alteration in blood flow despite 10 000 IU. None of the elective prevention group had further bleeding and obliteration was observed in 4/5 (80 %). A range of 600 to 10 000 IU of thrombin was used and there were no adverse procedure-related outcomes. CONCLUSIONS: Our results are promising and suggest that EUS-guided thrombin injection may have a role in managing bleeding from gastric fundal varices.

Cyclols Revisited: Facile Synthesis of Medium-Sized Cyclic Peptides.

Medium-sized rings, particularly the corresponding cyclic peptides, are challenging synthetic targets. In the present study, we report an approach to medium-sized cyclic peptides through targeted formation and collapse of cyclol intermediates. This methodology operates on ß-amino imides derived from 2,5-diketopiperazines and offers a straightforward transition from frequently examined scaffolds in drug discovery to a rarely visited class of medium-sized rings.

An approach to acute lower gastrointestinal bleeding.

Lower gastrointestinal bleeding (LGIB) is a common problem that can be treated via a number of endoscopic, radiological and surgical approaches. Although traditionally managed by the colorectal surgeons, surgery should be considered a last resort given the variety of endoscopic and radiological approaches available. This article provides an overview on the common causes of acute LGIB and the various techniques at our disposal to control it.

Does the presence of a trainee compromise success of biliary cannulation at ERCP?

BACKGROUND AND STUDY AIMS: Findings in the literature are conflicting on whether trainee involvement in endoscopic retrograde cholangiopancreatography (ERCP) procedures is detrimental to cannulation success rates. We addressed this in a prospective study, where cannulation success with or without trainee presence was the primary outcome measure. PATIENTS AND METHODS: We prospectively recorded data on 2 senior endoscopists and their trainees over an 18-month period for ERCPs in patients with a virgin ampulla. Presence or absence of a trainee at ERCP procedures was pragmatic, reflecting their other service or training commitments or annual leave. For trainee presence, the training protocol allowed them 6 minutes of supervised time in which to achieve biliary cannulation after reaching the ampulla. Study outcome measures included cannulation success, time to cannulation, technique, whether this was achieved independently by the trainee, and complications. RESULTS: There were 219 procedures recorded and analyzed (134 with a trainee, 85 without). Three trainees were involved. Selective biliary cannulation was achieved in 201 (92 %) of cases. When a trainee was present, cannulation was successful in 122/134 procedures (91 %), compared to 79/85 (93 %) with a senior endoscopist alone ( P  = 0.8, Fisher's exact test). Mean time to biliary cannulation with a trainee present was 7 minutes, compared with 5 minutes with no trainee. Mean time for successful independent cannulation by the trainee was 4 minutes, and 9 minutes for a consultant following a trainee's attempt. There were no serious adverse events. CONCLUSION: Our study shows that with this training protocol, involvement of a trainee on a routine secondary care ERCP list does not impair biliary cannulation success, and does not prolong a subsequent attempt by the senior endoscopist if initially unsuccessful. These findings support the involvement of trainees in routine ERCP lists with this training protocol.

Oxadiazole grafts in peptide macrocycles.

Synthetic methods that provide control over macrocycle conformation and, at the same time, mitigate the polarity of peptide bonds represent valuable tools for the discovery of new bioactive molecules. Here, we report a macrocyclization reaction between a linear peptide, an aldehyde and (N-isocyanimino)triphenylphosphorane. This process generates head-to-tail cyclic peptidomimetics in a single step. This method is tolerant to variation in the peptide and aldehyde components and has been applied for the synthesis of 15-, 18-, 21- and 24-membered rings. The resulting peptide macrocycles feature a 1,3,4-oxadiazole and a tertiary amine in their scaffolds. This non-canonical backbone region acts as an endocyclic control element that promotes and stabilizes a unique intramolecular hydrogen-bond network and can lead to macrocycles with conformationally rigid turn structures. Oxadiazole-containing macrocycles can also display a high passive membrane permeability, an important property for the development of bioavailable peptide-based therapeutics.

Side-chain-to-tail cyclization of ribosomally derived peptides promoted by aryl and alkyl amino-functionalized unnatural amino acids.

A strategy for the production of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Two genetically encodable unnatural amino acids, bearing either an aryl or alkyl amino group, were investigated for their efficiency toward promoting the formation of medium to large-sized peptide macrocycles via intein-mediated side-chain-to-C-terminus cyclization. While only partial cyclization was observed with precursor proteins containing para-amino-phenylalanine, efficient peptide macrocyclization could be achieved using O-2-aminoethyl-tyrosine as the reactive moiety. Conveniently, the latter was generated upon quantitative, post-translational reduction of the azido-containing counterpart, O-2-azidoethyl-tyrosine, directly in E. coli cells. This methodology could be successfully applied for the production of a 12 mer cyclic peptide with enhanced binding affinity for the model target protein streptavidin as compared to the acyclic counterpart (KD: 5.1 µM vs. 22.4 µM), thus demonstrating its utility toward the creation and investigation of novel, functional macrocyclic peptides.

Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Aminothiol Unnatural Amino Acids.

A versatile method for orchestrating the formation of side chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low-micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in Escherichia coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles.

Social values as arguments: similar is convincing.

Politicians, philosophers, and rhetors engage in co-value argumentation: appealing to one value in order to support another value (e.g., "equality leads to freedom"). Across four experiments in the United Kingdom and India, we found that the psychological relatedness of values affects the persuasiveness of the arguments that bind them. Experiment 1 found that participants were more persuaded by arguments citing values that fulfilled similar motives than by arguments citing opposing values. Experiments 2 and 3 replicated this result using a wider variety of values, while finding that the effect is stronger among people higher in need for cognition and that the effect is mediated by the greater plausibility of co-value arguments that link motivationally compatible values. Experiment 4 extended the effect to real-world arguments taken from political propaganda and replicated the mediating effect of argument plausibility. The findings highlight the importance of value relatedness in argument persuasiveness.

Designer macrocyclic organo-peptide hybrids inhibit the interaction between p53 and HDM2/X by accommodating a functional α-helix.

We report the design of side-chain-to-tail linked organo-peptide hybrids incorporating an α-helical protein-binding motif. Using this strategy, macrocyclic inhibitors of the p53:HDM2 interaction displaying dual specificity against the HDMX homolog as well as increased proteolytic stability could be obtained.

Use of the visual range of detection to estimate effective sweep width for land search and rescue based on 10 detection experiments in North America.

OBJECTIVE: Standard-of-practice search management requires that the probability of detection (POD) be determined for each search resource after a task. To calculate the POD, a detection index (W) is obtained by field experiments. Because of the complexities of the land environment, search planners need a way to estimate the value of W without conducting formal experiments. We demonstrate a robust empirical correlation between detection range (Rd) and W, and argue that Rd may reliably be used as a quick field estimate for W. METHODS: We obtained the average maximum detection range (AMDR), Rd, and W values from 10 detection experiments conducted throughout North America. We measured the correlation between Rd and W, and tested whether the apparent relationship between W and Rd was statistically significant. RESULTS: On average we found W ≈ 1.645 × Rd with a strong correlation (R(2) = .827). The high-visibility class had W ≈ 1.773 × Rd (also R(2) = .867), the medium-visibility class had W ≈ 1.556 × Rd (R(2) = .560), and the low-visibility had a correction factor of 1.135 (R(2) = .319) for Rd to W. Using analysis of variance and post hoc testing, only the high- and low-visibility classes were significantly different from each other (P < .01). We also found a high correlation between the AMDR and Rd (R(2) = .9974). CONCLUSIONS: Although additional experiments are required for the medium- and low-visibility search objects and in the dry-domain ecoregion, we suggest search planners use the following correction factors to convert field-measured Rd to an estimate of the effective sweep width (W): high-visibility W = 1.8 × Rd; medium-visibility W = 1.6 × Rd; and low-visibility W = 1.1 × Rd.

Design, synthesis, and diversification of ribosomally derived peptide macrocycles.

Ring topologies are widespread structural motifs among biologically active peptides found in nature. The recurrence of this motif is linked to the inherent advantages resulting from backbone cyclization, which include increased resistance against proteolytic degradation, improved cell permeability, and tighter and more specific interaction with the respective biomolecular target. Inspired by these natural product topologies, a number of groups have recently focused on developing methodologies that hinge upon the chemical elaboration of ribosomally derived polypeptides toward the synthesis and diversification of macrocyclic peptide structures. In this review, we highlight recent advances in this emerging new area and discuss the opportunities created by these methods toward the discovery of new functional entities.