Stapled Phd Peptides Inhibit Doc Toxin Induced Growth Arrest in Salmonella.

Bacterial toxin inhibition is a promising approach to overcoming antibiotic failure. InSalmonella, knockout of the toxin Doc has been shown to significantly reduce the formation of antibiotic-tolerant persisters. Doc is a kinase that is inhibited in nontolerant cells by its cognate antitoxin, Phd. In this work, we have developed first-in-class stapled peptide antitoxin mimetics based on the Doc inhibitory sequence of Phd. After making a series of substitutions to improve bacterial uptake, we identified a lead stapled Phd peptide that is able to counteract Doc toxicity in Salmonella. This provides an exciting starting point for the further development of therapeutic peptides capable of reducing antibiotic persistence in pathogenic bacteria.

Aromatic oligoesters as novel helix mimetic scaffolds.

The design, synthesis, and conformational analysis of a novel aromatic oligoester helix mimetic scaffold is reported. A range of amino acid-type side-chain functionality can be readily incorporated into monomer building blocks over three facile synthetic steps. Analysis of representative dimers revealed a stable conformer capable of effective mimicry of a canonical α-helix and the scaffold was found to be surprisingly stable to degradation in aqueous solutions at acidic and neutral pH.

In Situ Simulation Training for Frailty.

BACKGROUND: People living with frailty account for a significant proportion of hospital inpatients and are at increased risk of adverse events during admission. The understanding of frailty remains variable among hospital staff, and there is a need for effective frailty training across multidisciplinary teams. Simulation is known to be advantageous for improving human factor skills in multidisciplinary teams. In situ simulation can increase accessibility and promote ward team learning, but its effectiveness with respect to frailty has not been explored. METHOD: A single-centre, multi-fidelity, inter-professional in situ frailty simulation programme was developed. One-hour sessions were delivered weekly using frailty-based clinical scenarios. Mixed-method evaluation was used, with data collected pre- and post-session for comparison. RESULTS: In total, 86 multidisciplinary participants attended 19 sessions. There were significant improvements in self-efficacy rating across 10 of 12 human factor domains and in all frailty domains (p < 0.05). The common learning themes were situational awareness, communication and teamwork. Participants commented on the value of learning within ward teams and having the opportunity to debrief. CONCLUSION: In situ simulation can improve the self-efficacy of clinical and human factor skills related to frailty. The results are limited by the nature of self-reporting methods, and further studies assessing behavioural change and clinical outcomes are warranted.

A novel class of sulphonamides potently block malaria transmission by targeting a Plasmodium vacuole membrane protein.

Phenotypic cell-based screens are critical tools for discovering candidate drugs for development, yet identification of the cellular target and mode of action of a candidate drug is often lacking. Using an imaging-based screen, we recently discovered an N-[(4-hydroxychroman-4-yl)methyl]-sulphonamide (N-4HCS) compound, DDD01035881, that blocks male gamete formation in the malaria parasite life cycle and subsequent transmission of the parasite to the mosquito with nanomolar activity. To identify the target(s) of DDD01035881, and of the N-4HCS class of compounds more broadly, we synthesised a photoactivatable derivative, probe 2. Photoaffinity labelling of probe 2 coupled with mass spectrometry identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as a potential parasite target. Complementary methods including cellular thermal shift assays confirmed that the parent molecule DDD01035881 stabilised Pfs16 in lysates from activated mature gametocytes. Combined with high-resolution, fluorescence and electron microscopy data, which demonstrated that parasites inhibited with N-4HCS compounds phenocopy the targeted deletion of Pfs16 in gametocytes, these data implicate Pfs16 as a likely target of DDD01035881. This finding establishes N-4HCS compounds as being flexible and effective starting candidates from which transmission-blocking antimalarials can be developed in the future.

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in Salmonella.

In the search for novel antimicrobial therapeutics, toxin-antitoxin (TA) modules are promising yet underexplored targets for overcoming antibiotic failure. The bacterial toxin Doc has been associated with the persistence of Salmonella in macrophages, enabling its survival upon antibiotic exposure. After developing a novel method to produce the recombinant toxin, we have used antitoxin-mimicking peptides to thoroughly investigate the mechanism by which its cognate antitoxin Phd neutralizes the activity of Doc. We reveal insights into the molecular detail of the Phd-Doc relationship and discriminate antitoxin residues that stabilize the TA complex from those essential for inhibiting the activity of the toxin. Coexpression of Doc and antitoxin peptides in Salmonella was able to counteract the activity of the toxin, confirming our in vitro results with equivalent sequences. Our findings provide key principles for the development of chemical tools to study and therapeutically interrogate this important class of protein-protein interactions.

Modulators of protein-protein interactions as antimicrobial agents.

Protein-Protein interactions (PPIs) are involved in a myriad of cellular processes in all living organisms and the modulation of PPIs is already under investigation for the development of new drugs targeting cancers, autoimmune diseases and viruses. PPIs are also involved in the regulation of vital functions in bacteria and, therefore, targeting bacterial PPIs offers an attractive strategy for the development of antibiotics with novel modes of action. The latter are urgently needed to tackle multidrug-resistant and multidrug-tolerant bacteria. In this review, we describe recent developments in the modulation of PPIs in pathogenic bacteria for antibiotic development, including advanced small molecule and peptide inhibitors acting on bacterial PPIs involved in division, replication and transcription, outer membrane protein biogenesis, with an additional focus on toxin-antitoxin systems as upcoming drug targets.

What you need to know about falls.

Falls are a common presenting complaint, particularly in older patients, and are associated with significant morbidity. Inpatient falls also have financial implications for healthcare systems, including litigation costs. This article provides an approach to assessing a patient presenting with a fall, encompassing the cause and consequence of the event. It also highlights the need to consider both the acute and chronic factors that predispose a particular patient to fall. Chronic factors such as frailty, sarcopenia, cognitive impairment, and continence issues are often under-recognised and, as a result, not managed optimally. A comprehensive geriatric assessment is an ideal structure to identify modifiable risks. Practical interventions that can be of benefit to minimise a patient's risk of falling include a medication review, assessment of their mobility and their environment. In addition, continence review and visual assessment may be appropriate.

A randomized controlled trial of real versus sham acupuncture for basal thumb joint arthritis.

We report a single-blinded randomized controlled trial comparing acupuncture to sham (non-penetrating) needling for relief of symptoms of basal thumb joint arthritis. Seventy acupuncture naive patients with basal thumb joint arthritis were randomized to receive true acupuncture or sham needling with 35 patients in each arm. Blinded baseline and post-treatment assessments included visual analogue pain scores for different grips and movement. Function was assessed using the Nelson questionnaire. Both groups showed statistically and clinically significant improvements in pain at week one post-treatment compared with baseline, but there was no difference between the treatment groups. The pain relief was comparable with published data for some standard treatments. Acupuncture did not perform better than sham needling in this study, indicating that pain relief may have been achieved through non-specific mechanisms. Level of evidence: I.

Structure-Activity Relationship Studies of a Novel Class of Transmission Blocking Antimalarials Targeting Male Gametes.

Malaria is still a leading cause of mortality among children in the developing world, and despite the immense progress made in reducing the global burden, further efforts are needed if eradication is to be achieved. In this context, targeting transmission is widely recognized as a necessary intervention toward that goal. After carrying out a screen to discover new transmission-blocking agents, herein we report our medicinal chemistry efforts to study the potential of the most robust hit, DDD01035881, as a male-gamete targeted compound. We reveal key structural features for the activity of this series and identify analogues with greater potency and improved metabolic stability. We believe this study lays the groundwork for further development of this series as a transmission blocking agent.

Thirsty work: Exploring children's experiences of preoperative fasting.

BACKGROUND: Preoperative fasting is performed to reduce aspiration risk during general anesthesia. Recommendations are that patients should fast for 6 hours from solids and nonhuman milk, 4 hours from breast milk, and 2 hours from clear fluids. However, previous studies have shown that children fast far in excess of these times, which can result in perioperative complications and unnecessary discomfort for the child. AIMS: This prospective, mixed-methods study aims to explore the experiences of pediatric patients undergoing preoperative fasting in Leeds General Infirmary. It also aims to investigate fasting durations of these patients and factors which influence these. METHODS: Over 2 weeks, surveys were distributed to all parents of elective pediatric patients and completed prior to their child being called to theater. Children over the age of six were offered a child survey, which had been specifically developed for the study, with visual Likert scales and an area for free text. The gathering of children's comments about their experience of preoperative fasting is unique to this study. RESULTS: Seventy-one parent surveys and 48 child surveys were completed, with a mean patient age of 8.3 years (SD 4.1). The mean preoperative fasting time for food was 11.7 hours (SD 4.4) and 6.9 hours (SD 5.0) for fluids. Fasting times were far in excess of the minimums recommended, negatively impacting patient experience with 34% reporting being hungry/very hungry and 19% thirsty/very thirsty. Most children's comments suggested that they coped well with the fasting; however, several children reported feelings of sadness and anxiety. CONCLUSION: Preoperative fasting times in pediatric patients far exceed the durations set by international guidelines. Given that many children reported extreme feelings of hunger and thirst or emotional effects from the fast, these durations need to be optimized in order to improve patient experience.

Design, Synthesis, and Conformational Analysis of Oligobenzanilides as Multifacial α-Helix Mimetics.

The design, synthesis, and conformational analysis of an oligobenzanilide helix mimetic scaffold capable of simultaneous mimicry of two faces of an α-helix is reported. The synthetic methodology provides access to diverse monomer building blocks amenable to solid-phase assembly in just four synthetic steps. The conformational flexibility of model dimers was investigated using a combination of solid and solution state methodologies supplemented with DFT calculations. The lack of noncovalent constraints allows for significant conformational plasticity in the scaffold, thus permitting it to successfully mimic residues i, i+2, i+4, i+6, i+7, and i+9 of a canonical α-helix.

A high throughput screen for next-generation leads targeting malaria parasite transmission.

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2-1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male-female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population.

Interfacing native and non-native peptides: using Affimers to recognise α-helix mimicking foldamers.

Selection methods are used to identify Affimers that recognise α-helix mimicking N-alkylated aromatic oligoamides thus demonstrating foldamer and natural α-amino acid codes are compatible.

Probing Protein Surfaces: QSAR Analysis with Helix Mimetics.

α-Helix-mediated protein-protein interactions (PPIs) are important targets for small-molecule inhibition; however, generic approaches to inhibitor design are in their infancy and would benefit from QSAR analyses to rationalise the noncovalent basis of molecular recognition by designed ligands. Using a helix mimetic based on an oligoamide scaffold, we have exploited the power of a modular synthesis to access compounds that can readily be used to understand the noncovalent determinants of hDM2 recognition by this series of cell-active p53/hDM2 inhibitors.

Selective and potent proteomimetic inhibitors of intracellular protein-protein interactions.

Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed "proteomimetics", which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.

Selective and Potent Proteomimetic Inhibitors of Intracellular Protein-Protein Interactions.

Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed "proteomimetics", which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.

Multivalent helix mimetics for PPI-inhibition.

The exploitation of multivalent ligands for the inhibition of protein-protein interactions has not yet been explored as a supramolecular design strategy. This is despite the fact that protein-protein interactions typically occur within the context of multi-protein complexes and frequently exploit avidity effects or co-operative binding interactions to achieve high affinity interactions. In this paper we describe preliminary studies on the use of a multivalent N-alkylated aromatic oligoamide helix mimetic for inhibition of p53/hDM2 and establish that protein dimerisation is promoted, rather than enhanced binding resulting from a higher effective concentration of the ligand.

Orthogonal functionalisation of α-helix mimetics.

α-Helix mediated protein-protein interactions are of major therapeutic importance. As such, the design of inhibitors of this class of interaction is of significant interest. We present methodology to modify N-alkylated aromatic oligoamide α-helix mimetics using 'click' chemistry. The effect is shown to modulate the binding properties of a series of selective p53/hDM2 inhibitors.

Double-degradable responsive self-assembled multivalent arrays--temporary nanoscale recognition between dendrons and DNA.

This article reports self-assembling dendrons which bind DNA in a multivalent manner. The molecular design directly impacts on self-assembly which subsequently controls the way these multivalent nanostructures bind DNA--this can be simulated by multiscale modelling. Incorporation of an S-S linkage between the multivalent hydrophilic dendron and the hydrophobic units responsible for self-assembly allows these structures to undergo triggered reductive cleavage, with dithiothreitol (DTT) inducing controlled breakdown, enabling the release of bound DNA. As such, the high-affinity self-assembled multivalent binding is temporary. Furthermore, because the multivalent dendrons are constructed from esters, a second slow degradation step causes further breakdown of these structures. This two-step double-degradation mechanism converts a large self-assembling unit with high affinity for DNA into small units with no measurable binding affinity--demonstrating the advantage of self-assembled multivalency (SAMul) in achieving highly responsive nanoscale binding of biological targets.

Polyglycerol-based amphiphilic dendrons as potential siRNA carriers for in vivo applications.

The development of nonviral synthetic vectors for clinical application of gene therapy using siRNA transfection technology is of particular importance for treatment of human diseases, which is yet an unsolved challenge. By employing a rational design approach, we have synthesized a set of well-defined, low-molecular-weight dendritic polyglycerol-based amphiphiles, which are decorated peripherally with the DAPMA (N,N-di-(3-aminopropyl)-N-(methyl)amine) moiety. The main differences that were introduced in the structural motif relate to dendron generation and the type of linker between the hydrophilic and hydrophobic segment. The synthesized amphiphiles were then characterized for their aggregation behaviour and further evaluated with respect to their siRNA transfection potential by comparing their physico-chemical and biological features. Our findings demonstrated that all four synthesized amphiphiles yielded high gene binding affinities. Furthermore, the ester-linked compounds (G1-Ester-DAPMA, G2-Ester-DAPMA) revealed noticeable gene silencing in vitro without affecting the cell viability in the tumor cell line 786-O. Remarkably, neither G1-Ester-DAPMA nor G2-Ester-DAPMA induced inflammatory side effects after systemic administration in vivo, which is noteworthy because such highly positively charged compounds are typically associated with toxicity concerns which in turn supports their prospective application for in vivo purposes. Therefore, we believe that these structures may serve as new promising alternatives for nonviral siRNA delivery systems and have great potential for further synthetic modifications.