Novel WRN Helicase Inhibitors Selectively Target Microsatellite-Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology. See related commentary by Wainberg, p. 1369.

Supporting Emergency Care Delivery Through Updated Emergency Nurse Practitioner Competencies.

As multidisciplinary emergency care becomes increasingly complex, all team members must be aware of their respective roles and responsibilities. In the emergency department, nurse practitioners are integral members of the team. They possess a wide range of clinical and leadership competencies that allow them to perform specific and differentiated tasks within the emergency department. A well-defined competency not only contributes to the promotion of a positive work culture but also clarifies performance expectations, identifies skill gaps, and supports team development. Furthermore, it allows the nurse practitioner to adapt to changing conditions while maintaining patient safety. The competencies of emergency nurse practitioners have evolved over the past 2 decades. The authors discuss the importance of establishing clear expectations for emergency nurse practitioner practice in this article and the alignment of competencies with organizational culture and objectives.

Photoaffinity labelling displacement assay using multiple recombinant protein domains.

The development and optimisation of a photoaffinity labelling (PAL) displacement assay is presented, where a highly efficient PAL probe was used to report on the relative binding affinities of compounds to specific binding sites in multiple recombinant protein domains in tandem. The N- and C-terminal bromodomains of BRD4 were used as example target proteins. A test set of 264 compounds annotated with activity against the bromodomain and extra-terminal domain (BET) family in ChEMBL were used to benchmark the assay. The pIC50 values obtained from the assay correlated well with orthogonal TR-FRET data, highlighting the potential of this highly accessible PAL biochemical screening platform.

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry.

The screening of covalent or 'reactive' fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light.

Efficient Ligand Discovery Using Sulfur(VI) Fluoride Reactive Fragments.

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors.

Interprofessional team-based education: A comparison of in-person and online learner experiences by method of delivery and health profession.

BACKGROUND: Building capacity for teamwork, communication, role clarification and recognition of shared values is essential for interprofessional healthcare workforce development. Requirements to demonstrate interprofessional practice competencies have coincided with pivots to online delivery. Comparison of in-person and online delivery models for interprofessional education is important for future curriculum design. PURPOSE: This article presents an evaluation of in-person and online delivery modes for interprofessional team-based education and compares learner experiences across different health professions. METHODS: Students from 13 health professions (n = 2236) participated between Spring 2020 and Fall 2021. In-person and online delivery models were compared, assessing learner perceptions of efficacy for interprofessional practice, using reflective pre-post responses to the Interprofessional Collaborative Competency Attainment Scale (ICCAS). RESULTS: Mean ICCAS scores improved for in-person and online delivery (0.79 vs 0.66), with strong effect (Cohen's D 2.03 and 1.31 respectively; p < 0.001). Statistically significant differences were observed across professions, although all experienced ICCAS score improvements. Logistical benefits were evident for online delivery. CONCLUSION: In-person and online interprofessional team-based education can provide valuable learner experiences for large student cohorts from multiple professions. ICCAS score differences should be weighed against potential logistical benefits of online delivery. Timing of delivery and determinants of differences in student response across professions warrant evaluation for future curriculum design.

Current Practice and Practice Competencies of Clinical Nurse Specialists Working in US Emergency Care Settings: A Survey Study.

PURPOSE/AIMS: The aim of this study was to investigate the current practice of clinical nurse specialists working in US emergency care settings to (1) explicate the application of the Emergency Nurses Association core competencies and define the specialized clinical nurse specialist role in emergency care and (2) align current clinical nurse specialist practice in emergency settings with the National Association of Clinical Nurse Specialists core competencies and the identified substantive areas of clinical nurse specialist practice. DESIGN: This study used a quantitative exploratory descriptive approach using survey data. METHODS: A purposive convenience sample was recruited from the Emergency Nurses Association and the National Association of Clinical Nurse Specialists. Participants completed a 39-item survey based on a consensus process to develop competencies for emergency department (ED)-situated clinical nurse specialists. RESULTS: Respondents (n = 285) reported spending more than 50% of their work time in a primary clinical nurse specialist role. Significant differences in practice were found between geographic location, setting, educational preparation, title protection status, and type of institution. CONCLUSIONS: Our findings suggest that that the competencies ascribed to ED-situated clinical nurse specialists are valid in both frequency and importance. However, ED-situated clinical nurse specialists are not fully credentialed or practicing to the full extent of their education and licenses, because of professional, legislative, and environmental limitations.

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.

MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine.

α-ketoglutarate (αKG) is a central metabolic node with a broad influence on cellular physiology. The αKG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other αKG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving compound stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that, under our experimental conditions, glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.

A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines 'direct-to-biology' high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

Hematoma Block: An Effective Alternative for Pain Management and Procedural Sedation.

Management of traumatic extremity injuries in the emergency department is typically time consuming, often requiring multiple resources including pain control and procedural sedation. A hematoma block is a safe and effective alternative treatment of pain and may eliminate the need for procedural sedation. The hematoma block requires fewer emergency department resources and may decrease time and costs that are associated with procedural sedation and analgesia. This technique is easily performed by the emergency nurse practitioner, and complications are rare. This article presents the appropriate technique required to perform a hematoma block for pain control and fracture reduction in patients with closed traumatic injuries.

Viral Meningitis: A Pediatric Case Study.

Meningitis is a significant viral, bacterial, or fungal infection of the meninges that cover and protect the brain and the spinal cord. Symptoms of meningitis may present rapidly or develop gradually over a period of days, manifesting with common prodromal flu-like symptoms of headache, photophobia, fever, nuchal rigidity, myalgias, and fatigue. Character and significance of symptoms vary by patient age. Symptoms of infection may improve spontaneously or worsen, becoming potentially lethal. Early recognition and treatment of meningitis are crucial to prevent morbidity and mortality. The case reviewed in this article focuses on viral meningitis in a pediatric patient that may be unrecognized or underreported because of indistinct symptoms. Epidemiology, pathophysiology, presentation, assessment techniques, diagnostics, clinical management, and health promotion relevant to viral meningitis are presented.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

Angiotensin-Converting Enzyme Inhibitor Angioedema.

Angioedema from angiotensin-converting enzyme inhibitors (ACEIs) is a potential, emergent, and frightening problem that presents to the emergency department. This article focuses on angioedema caused by using ACEIs. The presentation, pathology, diagnostic testing, treatment, and patient education of angioedema are explored. This article explores using fresh frozen plasma as an initial approach to the treatment of ACEI angioedema.