Discovery and Optimization of Potent, Efficacious and Selective Inhibitors Targeting EGFR Exon20 Insertion Mutations.

Herein, we report the identification and optimization of a series of potent inhibitors of EGFR Exon20 insertions with significant selectivity over wild-type EGFR. A strategically designed HTS campaign, multiple iterations of structure-based drug design (SBDD), and tactical linker replacement led to a potent and wild-type selective series of molecules and ultimately the discovery of 36. Compound 36 is a potent and selective inhibitor of EGFR Exon20 insertions and has demonstrated encouraging efficacy in NSCLC EGFR CRISPR-engineered H2073 xenografts that carry an SVD Exon20 insertion and reduced efficacy in a H2073 wild-type EGFR xenograft model compared to CLN-081 (5), indicating that 36 may have lower EGFR wild-type associated toxicity.

AiZynth impact on medicinal chemistry practice at AstraZeneca.

AstraZeneca chemists have been using the AI retrosynthesis tool AiZynth for three years. In this article, we present seven examples of how medicinal chemists using AiZynth positively impacted their drug discovery programmes. These programmes run the gamut from early-stage hit confirmation to late-stage route optimisation efforts. We also discuss the different use cases for which AI retrosynthesis tools are best suited.

Discovery, Optimization, and Biological Evaluation of Arylpyridones as Cbl-b Inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.

Reduced failure rates associated with playing a new online game developed to support learning of core content in human systems physiology.

A digital mobile card-matching game called eFlip was developed to assist second-year undergraduate medical science students to learn core content and understand key associations in physiology. Our team developed customized content of core physiological associations, of increasing difficulty, for upload on a generic card-matching platform. The generic game was extended with add-ons coded to allow identification and access of student usage data for students who consented to have their game usage linked to academic performance such as final course marks and grades. Here, we describe the development of the eFlip game content, the student usage profile, and the game's impact on learning. Students were invited to use eFlip prior to and during the final examination period. Of those who used eFlip, 152 students consented to participate in a study of game use on learning performance outcomes. Within the students who consented, 74 played the game and 78 did not play the game (nonplayers). The mean course mark of the students who played the game [69.57%; 95% confidence level (CI) (67.22, 71.92)] was higher than that of the nonplayers [65.33%; 95% CI (62.67, 67.99)] (P = 0.02). Playing eFlip was also associated with reduced failure rates in students who played the game (1% failure rate) compared with the students who did not play the game (10% failure rate; P = 0.02). The number of games played by students peaked just prior to the course final examination. Overall, students who chose to play eFlip demonstrated improved grades that were associated with a higher probability of passing the physiology course.

Sex Differences in Photoprotective Responses to 1,25-Dihydroxyvitamin D3 in Mice Are Modulated by the Estrogen Receptor-ß.

Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-ß-/-) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-ß-/-/-- versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-ß-/- mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-ß-/- mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-ß-/-, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-ß.

Skeletal Muscle and the Maintenance of Vitamin D Status.

Vitamin D, unlike the micronutrients, vitamins A, E, and K, is largely obtained not from food, but by the action of solar ultraviolet (UV) light on its precursor, 7-dehydrocholesterol, in skin. With the decline in UV light intensity in winter, most skin production of vitamin D occurs in summer. Since no defined storage organ or tissue has been found for vitamin D, it has been assumed that an adequate vitamin D status in winter can only be maintained by oral supplementation. Skeletal muscle cells have now been shown to incorporate the vitamin D-binding protein (DBP) from blood into the cell cytoplasm where it binds to cytoplasmic actin. This intracellular DBP provides an array of specific binding sites for 25-hydroxyvitamin D (25(OH)D), which diffuses into the cell from the extracellular fluid. When intracellular DBP undergoes proteolytic breakdown, the bound 25(OH)D is then released and diffuses back into the blood. This uptake and release of 25(OH)D by muscle accounts for the very long half-life of this metabolite in the circulation. Since 25(OH)D concentration in the blood declines in winter, its cycling in and out of muscle cells appears to be upregulated. Parathyroid hormone is the most likely factor enhancing the repeated cycling of 25(OH)D between skeletal muscle and blood. This mechanism appears to have evolved to maintain an adequate vitamin D status in winter.

Evolution of the sheep coat: the impact of domestication on its structure and development.

Wild sheep and many primitive domesticated breeds have two coats: coarse hairs covering shorter, finer fibres. Both are shed annually. Exploitation of wool for apparel in the Bronze Age encouraged breeding for denser fleeces and continuously growing white fibres. The Merino is regarded as the culmination of this process. Archaeological discoveries, ancient images and parchment records portray this as an evolutionary progression, spanning millennia. However, examination of the fleeces from feral, two-coated and woolled sheep has revealed a ready facility of the follicle population to change from shedding to continuous growth and to revert from domesticated to primitive states. Modifications to coat structure, colour and composition have occurred in timeframes and to sheep population sizes that exclude the likelihood of variations arising from mutations and natural selection. The features are characteristic of the domestication phenotype: an assemblage of developmental, physiological, skeletal and hormonal modifications common to a wide variety of species under human control. The phenotypic similarities appeared to result from an accumulation of cryptic genetic changes early during vertebrate evolution. Because they did not affect fitness in the wild, the mutations were protected from adverse selection, becoming apparent only after exposure to a domestic environment. The neural crest, a transient embryonic cell population unique to vertebrates, has been implicated in the manifestations of the domesticated phenotype. This hypothesis is discussed with reference to the development of the wool follicle population and the particular roles of Notch pathway genes, culminating in the specific cell interactions that typify follicle initiation.

Changing medical students' attitudes to and knowledge of deafness: a mixed methods study.

BACKGROUND AND AIM: Communication with healthcare professionals is challenging for those with hearing loss. This study aimed to determine the impact dedicated deaf awareness training could have on medical student's attitudes to and knowledge of deafness, and to explore ways of incorporating deaf awareness training into the core undergraduate medical curriculum. METHODS: A validated questionnaire was used to measure attitudes to and knowledge of deafness in those taking an optional deaf awareness and basic sign language module for second year medical students compared to students who took another module. Previous students on this module were also contacted and asked to complete the same questionnaire. Focus groups with these students explored ways to incorporate deaf awareness training into the core undergraduate medical curriculum. RESULTS: After completing the module, students had a more positive attitude to deaf individuals (p < 0.001), and higher knowledge scores (p = 0.027) in comparison to the control group. Examination of data revealed a significant negative association between years since undertaking the module and attitudes score (r = - 0.29, p = 0.04, n = 51), with no significant association for knowledge score (r = 0.22, p = 0.11, n = 52). Focus groups suggested integrating deaf awareness training into existing undergraduate communication skills teaching, with the inclusion of deaf tutors. CONCLUSIONS: This study indicates that incorporating a specialist module on deafness can improve attitudes to and knowledge of deafness. Importantly, this effect decreases over time, demonstrating the need for refresher training amongst junior doctors.

A competency framework for clinical pharmacists and heart failure.

OBJECTIVES: Heart failure is an escalating 'pandemic' with malignant outcomes. Clinical pharmacist heart failure services have been developing for the past two decades. However, little clarity is available on the additional advanced knowledge, skills and experience needed for pharmacists to practice safely and competently. We aimed to provide an expert consensus on the minimum competencies necessary for clinical pharmacists to deliver appropriate care to patients with heart failure. METHODS: There were four methodological parts; (1) establishing a project group from experts in the field; (2) review of the literature, including existing pharmacy competency frameworks in other specialities and previous heart failure curricula from other professions; (3) consensus building, including developing, reviewing and adapting the contents of the framework; and (4) write-up and dissemination to widen the impact of the project. KEY FINDINGS: The final framework defines minimum competencies relevant to heart failure for four different potential levels of specialism: all pharmacists regardless of role (Stage 1); all patient-facing clinical pharmacists (Stage 2); clinical pharmacists with specific planned roles in the care of heart failure patients (Stage 3); and regionally/nationally/internationally recognised expert pharmacists with a direct specialism in heart failure (Stage 4). CONCLUSIONS: The framework delivers the vital first step needed to help standardise care, give pharmacists a blueprint for career progression and continuing professional development and bring clarity to the role of the pharmacist. Future collaboration between professional bodies and training providers is needed to develop structured programmes to align with the framework and facilitate training and resultant accreditation.

Content and discontent: a qualitative exploration of obstacles to elearning engagement in medical students.

BACKGROUND: Elearning is ubiquitous in healthcare professions education. Its equivalence to 'traditional' educational delivery methods is well established. There is a research imperative to clarify when and how to use elearning most effectively to mitigate the potential of it becoming merely a 'disruptive technology.' Research has begun to broadly identify challenges encountered by elearning users. In this study, we explore in depth the perceived obstacles to elearning engagement amongst medical students. Sensitising concepts of achievement emotions and the cognitive demands of multi-tasking highlight why students' deeply emotional responses to elearning may be so important in their learning. METHODS: This study used focus groups as a data collection tool. A purposeful sample of 31 participated. Iterative data gathering and analysis phases employed a constant comparative approach to generate themes firmly grounded in participant experience. RESULTS: Key themes that emerged from the data included a sense of injustice, passivity and a feeling of being 'lost at sea'. The actual content of the elearning resource provided important context. CONCLUSIONS: The identified themes have strong emotional foundations. These responses, interpreted through the lens of achievement emotions, have not previously been described. Appreciation of their importance is of benefit to educators involved in curriculum development or delivery.

The Australian Longitudinal Study on Women's Health: Using Focus Groups to Inform Recruitment.

BACKGROUND: Recruitment and retention of participants to large-scale, longitudinal studies can be a challenge, particularly when trying to target young women. Qualitative inquiries with members of the target population can prove valuable in assisting with the development of effective recruiting techniques. Researchers in the current study made use of focus group methodology to identify how to encourage young women aged 18-23 to participate in a national cohort online survey. OBJECTIVE: Our objectives were to gain insight into how to encourage young women to participate in a large-scale, longitudinal health survey, as well as to evaluate the survey instrument and mode of administration. METHODS: The Australian Longitudinal Study on Women's Health used focus group methodology to learn how to encourage young women to participate in a large-scale, longitudinal Web-based health survey and to evaluate the survey instrument and mode of administration. Nineteen groups, involving 75 women aged 18-23 years, were held in remote, regional, and urban areas of New South Wales and Queensland. RESULTS: Focus groups were held in 2 stages, with discussions lasting from 19 minutes to over 1 hour. The focus groups allowed concord to be reached regarding survey promotion using social media, why personal information was needed, strategies to ensure confidentiality, how best to ask sensitive questions, and survey design for ease of completion. Recruitment into the focus groups proved difficult: the groups varied in size between 1 and 8 participants, with the majority conducted with 2 participants. CONCLUSIONS: Intense recruitment efforts and variation in final focus group numbers highlights the "hard to reach" character of young women. However, the benefits of conducting focus group discussions as a preparatory stage to the recruitment of a large cohort for a longitudinal Web-based health survey were upheld.

CYP11A1 in skin: an alternative route to photoprotection by vitamin D compounds.

Topical 1,25-dihydroxyvitamin D (1,25D) and other vitamin D compounds have been shown to protect skin from damage by ultraviolet radiation (UVR) in a process that requires the vitamin D receptor. Yet, while mice which do not express the vitamin D receptor are more susceptible to photocarcinogenesis, mice unable to 1α-hydroxylate 25-hydroxyvitamin D to form 1,25D do not show increased susceptibility to UVR-induced skin tumors. A possible explanation is that an alternative pathway, which does not involve 1α-hydroxylation, may produce photoprotective compounds from vitamin D. The cholesterol side chain cleavage enzyme CYP11A1 is expressed in skin and produces 20-hydroxyvitamin D3 (20OHD) as a major product of vitamin D3. We examined whether topical 20OHD would affect UVR-induced DNA damage, inflammatory edema or immune suppression produced in Skh:hr1 mice. Photoprotection by 20OHD at 23 or 46pmol/cm(2) against cyclobutane pyrimidine dimers (DNA lesions) after UVR in mice was highly effective, up to 98±0.8%, (p<0.001) and comparable to that of 1,25D. Sunburn edema measured as skinfold thickness 24h after UVR was also significantly reduced by 20OHD (p<0.001). In studies of contact hypersensitivity (CHS), which is suppressed by UVR, topical application of 20OHD to mice protected against UVR-induced immunosuppression (p<0.05), similar to the effect of 1,25D at similar doses (46±0.6% protection with 20OHD, 44±0.5% with 1,25D). Both UVR-induced DNA damage and immunosuppression contribute to increased susceptibility to UVR-induced skin tumors. This study indicates a potentially anti-photocarcinogenic role of the naturally occurring vitamin D metabolite, 20OHD, which does not depend on 1α-hydroxylation for generation. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Protection from ultraviolet damage and photocarcinogenesis by vitamin D compounds.

Vitamin D is primarily produced by a photochemical reaction in skin, using the energy of ultraviolet B radiation. Ultraviolet radiation in sunlight is also responsible for several types of DNA damage, immunosuppression and photoaging. A number of adaptive responses are known to occur in skin to increasing UV exposure, including increased pigmentation, increased thickness of the cornified layer of skin and upregulation of DNA repair pathways. In addition to these known responses, there is now sufficient evidence to suggest that the local vitamin D system in skin, which includes local production of the active hormone, 1,25 dihydroxyvitamin D, together with metabolites of over-irradiation products, and vitamin D receptor(s), also provide an adaptive response to UV. The vitamin D system in skin reduces DNA damage, inflammation and photocarcinogenesis. Because vitamin D is made in skin, sun damage is less than it would be otherwise.

Safe moving and handling of patients: an interprofessional approach.

A gap in the medical undergraduate curriculum on safe moving and handling of patients was identified, and a project to enhance moving and handling education for undergraduates in various healthcare disciplines was undertaken. A team of nurses, doctors, physiotherapists and e-learning professionals developed a cross-discipline e-learning resource, piloted with medical and nursing students at Queen's University Belfast. One outcome of the project was the development of a deeper recognition of the common curriculum across healthcare disciplines.

Uptake of 25-hydroxyvitamin D by muscle and fat cells.

Vitamin D status, measured as serum 25-hydroxyvitamin D (25OHD) concentration, is determined by rates of input and of degradation. The half-life of 25OHD is surprisingly long for a steroid and much longer than its blood transporter, vitamin D binding protein. There is some evidence to suggest that vitamin D itself is stored in fat, whereas 25OHD concentrations are usually related to muscle-related parameters such as lean body mass and exercise. Both muscle and fat cells come from the mesenchymal cell lineage. We recently published evidence for net uptake of 25OHD into differentiated muscle cells, in a process that was megalin dependent, and speculated that this uptake might contribute to the extended half-life of 25OHD. Whether 25OHD is also taken up into cells of the adipocyte lineage is not clear. In the current study, we used the C2 muscle cell line as a source of myoblasts that were differentiated in culture to myotubes and 3T3-L1 pre-adipocytes that were differentiated into adipocytes in culture. We incubated the cells with trititated 25OHD and measured net uptake 4 and 16h afterwards. Differentiated myotubes took up labeled 25OHD in a time-dependent process to a far greater extent than myoblasts. In contrast, pre-adipocytes, but not differentiated adipocytes, accumulated labeled 25OHD in a time-dependent manner, though to a lesser extent than myotubes. Myotubes, but not myoblasts, showed megalin expression by immunohistochemistry. Pre-adipocytes, but not adipocytes, also showed expression of megalin. Since skeletal muscle consists mainly of differentiated muscle cells, while adipose tissue is mainly differentiated fat cells, it seems likely that muscle, but not fat tissue, provides a large extravascular pool through which 25OHD circulates and that this protects 25OHD from degradation. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Evidence that Notch and Delta expressions have a role in dermal condensate aggregation during wool follicle initiation.

Notch pathway genes have been implicated in the commitment of mesenchymal cells to a wool follicle cell fate. Notch1 and Delta1 transcripts were quantified in fetal skin of fine-woolled (Merino) and strong-woolled (Tukidale) sheep at two time points: either preceding (d56) or during (d70) the first wave of follicle initiation. DIG-labelled probes for both transcripts were localised in the epithelium, some mesenchymal cells, and in the dermal condensates of primordia. The possibility that condensates selectively incorporated Delta1-labelled mesenchymal cells is considered. The involvement of Notch1 in condensate formation was also explored in cultured fetal skin explants and whisker papilla cells using DAPT to block Notch signalling. In its presence, follicle initiation in skin explants was reduced, and the propensity for cultured papilla cells to aggregate was abolished. Results suggest that Notch1 activation is a prerequisite for mesenchymal aggregation. It is speculated that Delta interactions contribute to condensate formation, in vivo.

Vitamin D and death by sunshine.

Exposure to sunlight is the major cause of skin cancer. Ultraviolet radiation (UV) from the sun causes damage to DNA by direct absorption and can cause skin cell death. UV also causes production of reactive oxygen species that may interact with DNA to indirectly cause oxidative DNA damage. UV increases accumulation of p53 in skin cells, which upregulates repair genes but promotes death of irreparably damaged cells. A benefit of sunlight is vitamin D, which is formed following exposure of 7-dehydrocholesterol in skin cells to UV. The relatively inert vitamin D is metabolized to various biologically active compounds, including 1,25-dihydroxyvitamin D3. Therapeutic use of vitamin D compounds has proven beneficial in several cancer types, but more recently these compounds have been shown to prevent UV-induced cell death and DNA damage in human skin cells. Here, we discuss the effects of vitamin D compounds in skin cells that have been exposed to UV. Specifically, we examine the various signaling pathways involved in the vitamin D-induced protection of skin cells from UV.

1α,25-Dihydroxyvitamin D3 reduces several types of UV-induced DNA damage and contributes to photoprotection.

Vitamin D production requires UVB. In turn, we have shown that vitamin D compounds reduce UV-induced damage, including inflammation, sunburn, thymine dimers, the most frequent type of cyclobutane pyrimidine dimer, immunosuppression, and photocarcinogenesis. Our previous studies have shown most of the photoprotective effects by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) occurred through the nongenomic pathway because similar protection was seen with an analog, 1α,25-dihydroxylumistrol3 (JN), which has little ability to alter gene expression and also because a nongenomic antagonist of 1,25(OH)2D3 abolished protection. In the current study, we tested whether this photoprotective effect would extend to other types of DNA damage, and whether this could be demonstrated in human ex vivo skin, as this model would be suited to pre-clinical testing of topical formulations for photoprotection. In particular, using skin explants, we examined a time course for thymine dimers (TDs), the most abundant DNA photolesion, as well as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a mutagenic DNA base lesion arising from UV-induced oxidative stress, and 8-nitroguanosine (8-NG). Nitric oxide products, known markers for chronic inflammation and carcinogenesis, are also induced by UV. This study showed that 1,25(OH)2D3 significantly reduced TD and 8-NG as early as 30min post UV, and 8-oxodG at 3h post UV, confirming the photoprotective effect of 1,25(OH)2D3 against DNA photoproducts in human skin explants. At least in part, the mechanism of photoprotection by 1,25(OH)2D3 is likely to be through the reduction of reactive nitrogen species and the subsequent reduction in oxidative and nitrosative damage. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Opening of chloride channels by 1α,25-dihydroxyvitamin D3 contributes to photoprotection against UVR-induced thymine dimers in keratinocytes.

UVR produces vitamin D in skin, which is hydroxylated locally to 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). 1,25(OH)(2)D(3) protects skin cells against UVR-induced DNA damage, including thymine dimers, but the mechanism is unknown. As DNA repair is inhibited by nitric oxide (NO) products but facilitated by p53, we examined whether 1,25(OH)(2)D(3) altered the expression of nitrotyrosine, a product of NO, or p53 after UVR in human keratinocytes. 1,25(OH)(2)D(3) and the nongenomic agonist 1α,25-dihydroxylumisterol(3) reduced nitrotyrosine 16 hours after UVR, detected by a sensitive whole-cell ELISA. p53 was enhanced after UVR, and this was further augmented in the presence of 1,25(OH)(2)D(3). DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a chloride channel blocker previously shown to prevent 1,25(OH)(2)D(3)-induced chloride currents in osteoblasts, had no effect on thymine dimers on its own but prevented the 1,25(OH)(2)D(3)-induced protection against thymine dimers. Independent treatment with DIDS, at concentrations that had no effect on thymine dimers, blocked UVR-induced upregulation of p53. In contrast, reduction of nitrotyrosine remained in keratinocytes treated with 1,25(OH)(2)D(3) and DIDS at concentrations shown to block decreases in post-UVR thymine dimers. These results suggest that 1,25(OH)(2)D(3)-induced chloride currents help protect from UVR-induced thymine dimers, but further increases in p53 or reductions of nitrotyrosine by 1,25(OH)(2)D(3) are unlikely to contribute substantially to this protection.