Enhancing the Small-Scale Screenable Biological Space beyond Known Chemogenomics Libraries with Gray Chemical Matter─Compounds with Novel Mechanisms from High-Throughput Screening Profiles.

Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors.

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

Seasonal Accumulated Workloads in Collegiate Women's Soccer: A Comparison of Starters and Reserves.

Research quantifying the unique workload demands of starters and reserves in training and match settings throughout a season in collegiate soccer is limited. Purpose: The purpose of the current study is to compare accumulated workloads between starters and reserves in collegiate soccer. Methods: Twenty-two NCAA Division III female soccer athletes (height: 1.67 ± 0.05 m; body mass: 65.42 ± 6.33 kg; fat-free mass: 48.99 ± 3.81 kg; body fat %: 25.22 ± 4.78%) were equipped with wearable global positioning systems with on-board inertial sensors, which assessed a proprietary training load metric and distance covered for each practice and 22 matches throughout an entire season. Nine players were classified as starters (S), defined as those playing >50% of playing time throughout the entire season. The remaining 17 were reserves (R). Goalkeepers were excluded. A one-way ANOVA was used to determine the extent of differences in accumulated training load throughout the season by player status. Results: Accumulated training load and total distance covered for starters were greater than reserves ((S: 9431 ± 1471 vs. R: 6310 ± 2263 AU; p < 0.001) and (S: 401.7 ± 31.9 vs. R: 272.9 ± 51.4 km; p < 0.001), respectively) throughout the season. Conclusions: Starters covered a much greater distance throughout the season, resulting in almost double the training load compared to reserves. It is unknown if the high workloads experienced by starters or the low workloads of the reserves is more problematic. Managing player workloads in soccer may require attention to address potential imbalances that emerge between starters and reserves throughout a season.

Registered nurses' experience as disaster preparedness coordinators during a major incident: A qualitative study.

AIMS: To explore registered nurses' experiences as disaster preparedness coordinators of hospital incident command groups' during a major incident. DESIGN: A qualitative descriptive design using semi-structured interview. METHODS: This was a qualitative study based on one focus group discussion and six individual follow-up interviews. Participants were registered nurses in their capacity as disaster preparedness coordinators with experience from Major Incident simulations and a real-life Major Incident. The interviews were transcribed verbatim and analysed using content analysis. The COREQ checklist was used for reporting the findings. RESULTS: The analysis of data generated the main category: Expectations, previous experience and uncertainty affect hospital incident command group response during a Major Incident and three categories, (I) Gaining situational awareness (containing two subcategories), (II) Transitioning to management (containing three subcategories) and (III) Actions taken during uncertainty (containing two subcategories).

Chemoproteomics Enabled Discovery of Selective Probes for NuA4 Factor BRD8.

Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside a homology-model-guided design, to develop chemical biology approaches for the study of BRD8 inhibition and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

Emergency department registered nurses overestimate their disaster competency: A cross-sectional study.

BACKGROUND: Major incidents continue to pose a threat to health care systems by overwhelming them with a sudden surge of patients. A major factor impacting a hospital's surge capacity is the skills, abilities, and knowledge of emergency department (ED) registered nurses (RN). The level of disaster nursing competency they possess affects patient safety and outcome. ED RNs' ability to accurately assess their competency and knowledge is imperative for mitigating the effect of major incidents. ED RN's perception of overall disaster preparedness has not been thoroughly addressed. The aim of this study was to assess emergency department registered nurses' self-perceived disaster preparedness. METHOD: The study was a cross-sectional study per the STROBE checklist. A self-assessment questionnaire based on the results of a study identifying specific disaster nursing competencies for ED RNs was distributed to all ED RNs at six participating hospitals between January 10th to February 19th of 2019. A five-point Likert-type scale was used to assess competency. RESULTS: ED RNs' disaster preparedness according to the Total Disaster Competency mean was low. Furthermore, the results indicate that ED RNs' significantly overestimate their disaster nursing competency when compared to the Total Disaster Competency mean. Additionally, this study identified factors such as experience and education were positively associated with disaster preparedness and self-assessment ability. CONCLUSION: ED RNs' overestimate their disaster preparedness. However, ED RNs with experience and education may be better prepared. ED RNs with formal disaster education appeared to have better insight concerning their preparedness. Clinical experience, advanced levels of education, and training were positively associated with preparedness. Overestimating disaster competencies may negatively impact patient outcomes during a major incident.

Sevelamer crystals-an unusual cause of large bowel obstruction.

Sevelamer is a common phosphate binder used to manage hyperphosphatemia in end-stage renal disease. The medication has a well-documented gastrointestinal side-effect profile including nausea, vomiting and abdominal pain. There are few case reports of Sevelamer crystal deposition causing gastrointestinal mucosal injury, pseudotumor or obstruction. Here, we discuss a patient on Sevelamer who required operative management of a sigmoid obstruction. Surgical pathology showed pericolonic abscess with Sevelamer crystals.

Hospital incident command groups' performance during major incident simulations: a prospective observational study.

BACKGROUND: Hospital incident command groups' (HICG) performance may have a profound impact on hospital response to major incidents. Previous research has assessed hospital incident command group capacity as opposed to performance and factors associated to performance. The objective was to assess associations between decision-making and staff procedure skills of the hospital incident command group. METHODS: This was a prospective observational study using performance indicators to assess hospital incident command groups' decision-making and performance. A total of six hospitals in Stockholm, Sweden, with their respective HICGs participated. Associations between decision-making skills and staff procedure skills during major incident simulations were assessed using measurable performance indicators. RESULTS: Decision-making skills are correlated to staff procedure skills and overall HICG performance. Proactive decision-making skills had significantly lower means than reactive decision-making skills and are significantly correlated to staff procedure skills. CONCLUSION: There is a significant correlation between decision-making skills and staff procedural skills. Hospital incident command groups' proactive decision-making abilities tended to be less developed than reactive decision-making abilities. These proactive decision-making skills may be a predictive factor for overall hospital incident command group performance. A lack of proactive decision-making ability may hamper efforts to mitigate the effects of a major incident.

Match Demands of Women's Collegiate Soccer.

Research describing the match and specific positional demands during match play in women's collegiate soccer is limited. The purpose of the study was to quantify the match demands of National Collegiate Athletic Association (NCAA) Division III soccer and assess position differences in movement kinematics, heart rate (HR), and energy expenditure. Twenty-five Division III women soccer players (height: 1.61 ± 0.3 m; body mass: 66.7 ± 7.5 kg; fat-free mass: 50.3 ± 6.5 kg; body fat%: 25.6 ± 5.1%) were equipped with a wearable global positioning system to assess the demands of 22 matches throughout a season. Players were categorized by position (goal keepers (GK), center defenders (CB), flank players (FP), forwards (F), and center midfielders (CM)). Players covered 9807 ± 2588 m and 1019 ± 552 m at high speeds (>249.6 m·m-1), with an overall average speed of 62.85 ± 14.7 m·m-1. This resulted in a mean HR of 74.2 ± 6% HR max and energy expenditure of 1259 ± 309 kcal. Significant and meaningful differences in movement kinematics were observed across position groups. CM covered the most distance resulting in the highest training load. FP covered the most distance at high speeds and mean HR values were highest in CM, CB, and FP positions.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

A small-molecule inhibitor of C5 complement protein.

The complement pathway is an important part of the immune system, and uncontrolled activation is implicated in many diseases. The human complement component 5 protein (C5) is a validated drug target within the complement pathway, as an anti-C5 antibody (Soliris) is an approved therapy for paroxysmal nocturnal hemoglobinuria. Here, we report the identification, optimization and mechanism of action for the first small-molecule inhibitor of C5 complement protein.

Magnetic particles with perpendicular anisotropy for mechanical cancer cell destruction.

We demonstrate the effectiveness of out-of-plane magnetized magnetic microdiscs for cancer treatment through mechanical cell disruption under an applied rotating magnetic field. The magnetic particles are synthetic antiferromagnets formed from a repeated motif of ultrathin CoFeB/Pt layers. In-vitro studies on glioma cells are used to compare the efficiency of the CoFeB/Pt microdiscs with Py vortex microdiscs. It is found that the CoFeB/Pt microdiscs are able to damage 62 ± 3% of cancer cells compared with 12 ± 2% after applying a 10 kOe rotating field for one minute. The torques applied by each type of particle are measured and are shown to match values predicted by a simple Stoner-Wohlfarth anisotropy model, giving maximum values of 20 fNm for the CoFeB/Pt and 75 fNm for the Py vortex particles. The symmetry of the anisotropy is argued to be more important than the magnitude of the torque in causing effective cell destruction in these experiments. This work shows how future magnetic particles can be successfully designed for applications requiring control of applied torques.

Hitting a Moving Target: Glioma Stem Cells Demand New Approaches in Glioblastoma Therapy.

BACKGROUND: Glioblastoma multiforme (GBM) continues to devastate patients and outfox investigators and clinicians despite the preponderance of research directed at its biology, pathogenesis and therapeutic advances. GBM routinely outlasts multidisciplinary treatment protocols, almost inevitably recurring in a yet more aggressive and resistant form with distinct genetic differences from the original tumor. Attempts to glean further insight into GBM point increasingly toward a subpopulation of cells with a stem-like phenotype. These cancer stem cells, similar to those now described in a variety of malignancies, are capable of tumorigenesis from a population of susceptible cells. CONCLUSIONS: Glioma stem cells have thus become a prevalent focus in GBM research for their presumed role in development, maintenance and recurrence of tumors. Glioma stem cells infiltrate the white matter surrounding tumors and often evade resection. They are uniquely suited both biochemically and environmentally to resist the best therapy currently available, intrinsically and efficiently resistant to standard chemo- and radiotherapy. These stem cells create an extremely heterogenous tumor that to date has had an answer for every therapeutic question, with continued dismal patient survival. Targeting this population of glioma stem cells may hold the long-awaited key to durable therapeutic efficacy in GBM.

Evaluation of Disaster Preparedness Based on Simulation Exercises: A Comparison of Two Models.

OBJECTIVE: The objective of this study was to highlight 2 models, the Hospital Incident Command System (HICS) and the Disaster Management Indicator model (DiMI), for evaluating the in-hospital management of a disaster situation through simulation exercises. METHODS: Two disaster exercises, A and B, with similar scenarios were performed. Both exercises were evaluated with regard to actions, processes, and structures. After the exercises, the results were calculated and compared. RESULTS: In exercise A the HICS model indicated that 32% of the required positions for the immediate phase were taken under consideration with an average performance of 70%. For exercise B, the corresponding scores were 42% and 68%, respectively. According to the DiMI model, the results for exercise A were a score of 68% for management processes and 63% for management structure (staff skills). In B the results were 77% and 86%, respectively. CONCLUSIONS: Both models demonstrated acceptable results in relation to previous studies. More research in this area is needed to validate which of these methods best evaluates disaster preparedness based on simulation exercises or whether the methods are complementary and should therefore be used together. (Disaster Med Public Health Preparedness. 2016;10:544-548).

Examining the influence of specificity ligands and ATP-competitive ligands on the overall effectiveness of bivalent kinase inhibitors.

BACKGROUND: Identifying selective kinase inhibitors remains a major challenge. The design of bivalent inhibitors provides a rational strategy for accessing potent and selective inhibitors. While bivalent kinase inhibitors have been successfully designed, no comprehensive assessment of affinity and selectivity for a series of bivalent inhibitors has been performed. Here, we present an evaluation of the structure activity relationship for bivalent kinase inhibitors targeting ABL1. METHODS: Various SNAPtag constructs bearing different specificity ligands were expressed in vitro. Bivalent inhibitor formation was accomplished by synthesizing individual ATP-competitive kinase inhibitors containing a SNAPtag targeting moiety, enabling the spontaneous self-assembly of the bivalent inhibitor. Assembled bivalent inhibitors were incubated with K562 lysates, and then subjected to affinity enrichment using various ATP-competitive inhibitors immobilized to sepharose beads. Resulting eluents were analyzed using Tandem Mass Tag (TMT) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Relative binding affinity of the bivalent inhibitor was determined by calculating the concentration at which 50% of a given kinase remained bound to the affinity matrix. RESULTS: The profiling of three parental ATP-competitive inhibitors and nine SNAPtag conjugates led to the identification of 349 kinase proteins. In all cases, the bivalent inhibitors exhibited enhanced binding affinity and selectivity for ABL1 when compared to the parental compound conjugated to SNAPtag alone. While the rank order of binding affinity could be predicted by considering the binding affinities of the individual specificity ligands, the resulting affinity of the assembled bivalent inhibitor was not predictable. The results from this study suggest that as the potency of the ATP-competitive ligand increases, the contribution of the specificity ligand towards the overall binding affinity of the bivalent inhibitor decreases. However, the affinity of the specificity components in its interaction with the target is essential for achieving selectivity. CONCLUSION: Through comprehensive chemical proteomic profiling, this work provides the first insight into the influence of ATP-competitive and specificity ligands binding to their intended target on a proteome-wide scale. The resulting data suggest a subtle interplay between the ATP-competitive and specificity ligands that cannot be accounted for by considering the specificity or affinity of the individual components alone.

Conversion of a Single Polypharmacological Agent into Selective Bivalent Inhibitors of Intracellular Kinase Activity.

Loss-of-function studies are valuable for elucidating kinase function and the validation of new drug targets. While genetic techniques, such as RNAi and genetic knockouts, are highly specific and easy to implement, in many cases post-translational perturbation of kinase activity, specifically pharmacological inhibition, is preferable. However, due to the high degree of structural similarity between kinase active sites and the large size of the kinome, identification of pharmacological agents that are sufficiently selective to probe the function of a specific kinase of interest is challenging, and there is currently no systematic method for accomplishing this goal. Here, we present a modular chemical genetic strategy that uses antibody mimetics as highly selective targeting components of bivalent kinase inhibitors. We demonstrate that it is possible to confer high kinase selectivity to a promiscuous ATP-competitive inhibitor by tethering it to an antibody mimetic fused to the self-labeling protein SNAPtag. With this approach, a potent bivalent inhibitor of the tyrosine kinase Abl was generated. Profiling in complex cell lysates, with competition-based quantitative chemical proteomics, revealed that this bivalent inhibitor possesses greatly enhanced selectivity for its target, BCR-Abl, in K562 cells. Importantly, we show that both components of the bivalent inhibitor can be assembled in K562 cells to block the ability of BCR-Abl to phosphorylate a direct cellular substrate. Finally, we demonstrate the generality of using antibody mimetics as components of bivalent inhibitors by generating a reagent that is selective for the activated state of the serine/threonine kinase ERK2.

Gift from Nature: Cyclomarin†A Kills Mycobacteria and Malaria Parasites by Distinct Modes of Action.

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action.