Targeting Cytotoxic Agents through EGFR-Mediated Covalent Binding and Release.

A major drawback of cytotoxic chemotherapy is the lack of selectivity toward noncancerous cells. The targeted delivery of cytotoxic drugs to tumor cells is a longstanding goal in cancer research. We proposed that covalent inhibitors could be adapted to deliver cytotoxic agents, conjugated to the ß-position of the Michael acceptor, via an addition-elimination mechanism promoted by covalent binding. Studies on model systems showed that conjugated 5-fluorouracil (5FU) could be released upon thiol addition in relevant time scales. A series of covalent epidermal growth factor receptor (EGFR) inhibitors were synthesized as their 5FU derivatives. Achieving the desired release of 5FU was demonstrated to depend on the electronics and geometry of the compounds. Mass spectrometry and NMR studies demonstrated an anilinoquinazoline acrylate ester conjugate bound to EGFR with the release of 5FU. This work establishes that acrylates can be used to release conjugated molecules upon covalent binding to proteins and could be used to develop targeted therapeutics.

Social Cognitive Theory, Driving Cessation, and Alternative Transportation in Later Life.

Having viable alternative transportation options could help individuals stop driving when appropriate. This study employs the Social Cognitive Theory (SCT) to understand the barriers and facilitators of alternative transportation among a sample of adults aged 55 and older (N = 32). Using a daily transportation data collection app, MyAmble, the research team asked participants questions structured around environmental, individual, and behavioral factors as outlined in the SCT framework. Responses were analyzed using directed content analysis. Findings suggest a substantial reliance on motor vehicles and it was evident that many participants had never seriously considered what they would do if they could no longer drive. We posit that SCT principles may be applied to help older adults build self-efficacy to transition to driving cessation when needed.

A Survey of Resources and Nursing Workforce for Clinical Research Delivery in Paediatric Intensive Care Within the UK / Ireland.

Introduction: Clinical research within Paediatric Intensive Care (PICU) is necessary to reduce morbidity and mortality associated within this resource-intensive environment. With UK PICUs encouraged to be research-active there was a drive to understand how centres support research delivery. Aim: To identify the research workforce available within UK/Ireland PICUs to support clinical research delivery. Method: An electronic survey, endorsed by the Paediatric Critical Care Society (PCCS), was designed and reported in accordance with CHERRIES guidelines. The survey was distributed by email to all UK/Ireland Nurse Managers and Medical/ Nursing Research leads, aiming for one response per site during the period of April-June 2021. Only one response per site was included in analysis. Results: 44 responses were received, representing 24/30 UK/Ireland sites (80% response rate). Responses from n = 21/30 units are included (three excluded for insufficient data). 90% (n = 19/21) units were research active, although only 52% (n = 11) had permanent research roles funded within their staffing establishment. The majority of units (n = 18, 86%) had less than two WTE research nurses. Resources were felt to be sufficient for current research delivery by 43% of units (n = 9), but this confidence diminished to 19% (n = 4) when considering their ability to support future research. The top barriers to research conduct were insufficiently funded/unfunded studies (52%; n = 11), clinical staff too busy to support research activity (52%; n = 11) and short-term/fixed-term contracts for research staff (38%; n = 8). Conclusion: Despite the perceived importance of research and 90% of responding UK/Ireland PICUs being research active, the majority have limited resources to support research delivery. This has implications for their ability to participate in future multi-centre trials and opportunities to support the development of future medical/nursing clinical academics. Further work is required to identify optimum models of clinical research delivery.

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Critical care scientists: Role, training and future directions.

Critical care scientists are a little known but increasingly prominent group of professionals, included in both the government-run Modernising Scientific Careers initiative and 2019 Guidelines for the Provision of Intensive Care Services. This article outlines the role of critical care scientists, their training programme and potential future directions for the role. A wider appreciation and acknowledgement of the critical care scientist's role within the multi-disciplinary team will allow critical care units to fully understand the potential benefits that may be brought to patient care and service delivery.

Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML.

Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies.

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM.

Peptidoglycan (PG) is the main component of bacterial cell walls and the target for many antibiotics. PG biosynthesis is tightly coordinated with cell wall growth and turnover, and many of these control activities depend upon PASTA-domain containing eukaryotic-like serine/threonine protein kinases (PASTA-eSTK) that sense PG fragments. However, only a few PG biosynthetic enzymes are direct kinase substrates. Here, we identify the conserved ReoM protein as a novel PASTA-eSTK substrate in the Gram-positive pathogen Listeria monocytogenes. Our data show that the phosphorylation of ReoM is essential as it controls ClpCP-dependent proteolytic degradation of the essential enzyme MurA, which catalyses the first committed step in PG biosynthesis. We also identify ReoY as a second novel factor required for degradation of ClpCP substrates. Collectively, our data imply that the first committed step of PG biosynthesis is activated through control of ClpCP protease activity in response to signals of PG homeostasis imbalance.

Using Community-Based Participatory Research Strategies in Age-Friendly Communities to Solve Mobility Challenges.

The number of older adults is steadily increasing in the United States and across the globe. Aging is linked to an increased risk of disability. Disabilities that limit one or more major life activities such as seeing, hearing, walking, and motor skills impact a person's ability to drive a car. Low utilization of alternative transportation by older adults and people with disabilities may put them at risk for social isolation. Social isolation is associated with a variety of negative health outcomes. While communities are challenged to create available, acceptable, accessible, adaptable and affordable mobility options, there are widely held, inaccurate biases around older adults' abilities to contribute to the development and improvement of alternative transportation options. Gerontological social workers are well-positioned to address this bias. This paper presents a case study of a large metropolitan county in the Midwest where community-based participatory research (CBPR) strategies were used to engage older residents to support the development of alternative transportation options supporting the tenets of environmental justice.

Tuning the Binding Affinity and Selectivity of Perfluoroaryl-Stapled Peptides by Cysteine-Editing.

A growing number of approaches to "staple" α-helical peptides into a bioactive conformation using cysteine cross-linking are emerging. Here, the replacement of l-cysteine with "cysteine analogues" in combinations of different stereochemistry, side chain length and beta-carbon substitution, is explored to examine the influence that the thiol-containing residue(s) has on target protein binding affinity in a well-explored model system, p53-MDM2/MDMX, which is constituted by the interaction of the tumour suppressor protein p53 and proteins MDM2 and MDMX, which regulate p53 activity. In some cases, replacement of one or more l-cysteine residues afforded significant changes in the measured binding affinity and target selectivity of the peptide. Computationally constructed homology models indicate that some modifications, such as incorporating two d-cysteine residues, favourably alter the positions of key functional amino acid side chains, which is likely to cause changes in binding affinity, in agreement with measured surface plasmon resonance data.

Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach.

ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

Benzene Probes in Molecular Dynamics Simulations Reveal Novel Binding Sites for Ligand Design.

Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design.

Genetic association studies of the FOXP3 gene in Graves' disease and autoimmune Addison's disease in the United Kingdom population.

Regulatory T lymphocytes play a crucial role in modulating potentially self-reactive clones, and dysfunction of this cell type contributes to autoimmune disease. FOXP3 is a critical determinant of CD(4+)CD(25+)T regulatory (T(reg)) cell development and function. The aim of this study was to investigate whether genetic polymorphisms at the FOXP3 locus predispose to autoimmune endocrinopathies. Five single nucleotide polymorphisms (SNPs) and two microsatellite polymorphisms were genotyped in our Caucasian cohorts of 633 unrelated Graves' disease (GD) subjects, 104 autoimmune Addison's disease (AAD) subjects and 528 healthy controls. SNP genotyping was performed by either restriction enzyme digestion or by primer-extension-MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) assay. Microsatellites were analysed using fluorescent PCR. Case-control analysis was performed using chi(2) testing on contingency tables for allele frequency. Haplotype analysis was performed using the UNPHASED package. No evidence for disease association was found with any of the seven polymorphisms in either of the GD or AAD subjects as compared with controls (P = 0.26-0.94). Haplotype analysis found a weak evidence for the association of a minor haplotype with GD; this was not significant when corrected for multiple testing. This study has found no robust evidence that FOXP3 gene polymorphism contributes to the susceptibility to GD or AAD in the UK population.

Physiologic variability of single-voxel proton MR spectroscopic measurements at 3T.

BACKGROUND AND PURPOSE: Physiologic and scanner variability of proton MR spectroscopy (MRS) measurements can limit the detection of subtle metabolite fluctuations. We assessed the variability of such measurements at 3T and compared two methods to obtain absolute concentrations. METHODS: Variability over 14 days was assessed with short-echo, single-voxel proton MRS in 14 control subjects and in a phantom containing 50 mmol/L N-acetylaspartate (NAA). Spectra were analyzed by using LCModel, scaling factors determined with both the calibration phantom (CP), and water peak intensity (WP) methods. Relative (reflecting the systematic drift) and absolute variability (reflecting the magnitude of scanner variability) was determined. RESULTS: For the phantom, initial (49 +/- 1.7 mmol/L) and second measurements (50 +/- 1.6 mmol/L) showed similar results, with small variability (relative, -0.6 +/- 1.5 mmol/L; absolute, 1.1 +/- 1.1 mmol/L). Control subjects had no systematic difference between the two scans for any measurement. Absolute variabilities in the temporal lobe for total NAA (NAA+NAAG) were 13% (CP) and 11% (WP). The largest variability (29%) was found for glutamate-glutamine (29%) with the CP method, and for myo-inositol with the WP method (28%). Absolute variability was smaller for the frontal lobe measurements (total NAA 7% and overall 6-18% for CP; total NAA 6% and overall 5-19% for WP). No significant difference was observed between the two methods. CONCLUSION: Physiologic variability is the major source of measurement variability and accounts for 12% of the variability in temporal lobe total NAA. Therefore, total NAA variations must clearly exceed this before they can reliably be attributed to an effect of disease.

No association of the codon 55 methionine to valine polymorphism in the SUMO4 gene with Graves' disease.

OBJECTIVE: A functional polymorphism at codon 55 of the small ubiquitin-like modifier-4 (SUMO4) gene (methionine to valine; M55V) has recently been associated with type 1 diabetes mellitus (T1D). We aimed to establish whether this locus also contributes towards the genetic susceptibility to Graves' disease (GD) and autoimmune Addison's disease. DESIGN: A case-control analysis was performed using genomic DNA samples from 595 unrelated white GD subjects, 104 white autoimmune Addison's disease subjects and 467 healthy white control subjects. The SUMO4 M55V single nucleotide polymorphism (SNP) was genotyped using polymerase chain reaction (PCR) amplification followed by digestion with the restriction enzyme MseI. RESULTS: There was no association of the SUMO4 M55V alleles with either GD, thyroid-associated orbitopathy or autoimmune Addison's disease when compared to controls; P = 0.28, 0.46 and 0.91, respectively, by chi2 testing. CONCLUSION: We cannot confirm a generalized role for SUMO4 in autoimmune endocrinopathy. The SUMO4 codon 55 methionine to valine polymorphism may be exclusively associated with susceptibility to T1D, or the effect of the locus in GD and Addison's disease may be much less than that found in T1D patients.

Role of the CD40 locus in Graves' disease.

The genetic basis for Graves' disease remains largely unknown, but significant linkage to microsatellite markers on 20q11 suggests that this region harbors a susceptibility gene. One obvious candidate gene at this 20q11 locus is CD40, which encodes a B-cell-surface receptor that is involved in T-cell to B-cell signaling and is implicated in control of T-cell autoreactivity. In addition, an allele of a single nucleotide polymorphism (SNP) in the Kozak consensus sequence of the 5' untranslated region of CD40 exon 1 has been reported to show modest evidence for association with Graves' disease. We have investigated the role of this 5' untranslated region (5' UTR) in Graves' disease susceptibility in our cohort of 451 unrelated white subjects with Graves' disease and 446 healthy controls. The CD40 5'UTR SNP (C --> T, position -1) was polymerase chain reaction (PCR)amplified and genotyped using the restriction enzyme NcoI. The frequency of the C allele was 74.8% in Graves' probands compared to 75.1% in controls (not significant [NS]). We find no evidence to support allelic association with Graves' disease at this CD40 SNP, despite the adequate power of the study. We are unable to confirm a role for CD40 in Graves' disease pathogenesis in our U.K. population, however, further studies involving larger patient cohorts and a saturated SNP marker map are required to resolve this issue.

Mutational analysis of the FOXP3 gene and evidence for genetic heterogeneity in the immunodysregulation, polyendocrinopathy, enteropathy syndrome.

The immunodysregulation, polyendocrinopathy, enteropathy syndrome (IPEX), is a rare disorder of immune regulation resulting in multiple autoimmune disorders, which demonstrates X-linked recessive inheritance. The disease gene, FOXP3, was identified in 2001, and several mutations within this gene have since been described in patients with IPEX. We used linkage analysis, mutational screening of the FOXP3 gene, human leukocyte antigen typing, and analysis of X-chromosome inactivation to investigate 2 kindreds (21 subjects in total) with 4 male infants (3 now deceased) and 1 girl affected by IPEX. In 1 family a novel FOXP3 mutation was identified in the proband, with a single base deletion at codon 76 of exon 2, leading to a frameshift, which predicted a truncated protein product (108 residues vs. 431 in wild type). In the second family, the FOXP3 locus was excluded by recombination, and mutational analysis of the gene was negative. The affected girl from this family was shown to have human leukocyte antigen DR2 and DR6 alleles and random X-chromosome inactivation in peripheral blood mononuclear cells. Our analysis has elucidated the molecular basis of IPEX in one family and has, for the first time, provided evidence for an autosomal locus, suggesting genetic heterogeneity in this syndrome.

The deleted in colorectal carcinoma (DCC) gene 201 R --> G polymorphism: no evidence for genetic association with autoimmune disease.

The product of the deleted in colorectal carcinoma (DCC) gene has a role in apoptosis and is a positional candidate for IDDM6, the putative chromosome 18q12-q23 autoimmune disease locus. We hypothesised that a nonconservative substitution (DCC 201 R --> G; nucleotide (nt) 601 C --> G), located in an extracellular immunoglobulin-like domain of DCC, is an aetiological determinant of autoimmunity. We tested this hypothesis by genetically testing the nt 601 C --> G polymorphism for association with three autoimmune phenotypes in a large population-based case-control study. There was no evidence for association of DCC nt 601 C --> G with autoimmune disease in cohorts comprising 2253 subjects with rheumatoid arthritis, type I diabetes and Graves' disease, and 2225 control subjects, from New Zealand and the United Kingdom. Furthermore, using the transmission disequilibrium test, there was no significant evidence for biased transmission of the nt 601 C --> G polymorphism to probands within a 382 family type I diabetes affected sibpair cohort from the United Kingdom. Thus, the DCC 201 R --> G polymorphism does not appreciably influence risk of developing the autoimmune diseases tested.