Towards the design and synthesis of a p53 mutant Y220C rescue drug

The DNA damage response is an important barrier to tumorigenesis. Impairment of p53 function is crucial to tumorigenesis by allowing evasion of p53 dependent responses. The mechanisms involve either (i) missense mutations, (ii) partial abrogation of signaling pathways or effector molecules that regulate p53, (iii) epigenetic deregulation. The tyrosine to cysteine mutation, Y220C, in p53 is found in around 100,000 new cancer cases per annum. This mutation destabilizes the core domain by 4 kcal mol-1 and destabilizes p53 under physiological conditions. The large to small mutation results in the fusing of two shallow pockets to create an extended surface cleft that a number of different fragments bind. The small molecule PK083, 1-(-ethyl-9H-carbazol-3-yl)-N-methanamine, binds the mutant-specific crevice with a KD = 150 μM and raised the protein mutant’s half-life to over 15 minutes vs. 4 minutes in the absence of the ligand. This presents an ideal starting point towards the design of a p53 rescuing drug. A library of carbazoles was designed and synthesized, guided by SAR studies, crystallographic information and computational chemistry, with the aim of optimizing the structure toward a more potent PK083 analogue. Affinity gains were achieved by exploitation of direct fluorine-protein interactions between PK9255 (N-methyl-1-(9- (2,2,2-trifluoroethyl)-9H-carbazol-3-yl)methanamine), and the backbone carbonyls of Leu145 and Trp146 and the thiol of Cys220, resulting in a Kd = 28 μM. Further affinity gains were achieved through SAR studies targeting the proline-rich subsite II. Chemistry was optimized to allow a diversity-oriented synthesis toward 2,6,9- substituted carbazoles. A small library of PK083 analogues, where the subsite II targeting group was a halogen, ether, ester, amide or heterocycle were synthesized, identifying the heterocyclic compounds as most potent. A scan of heterocyclic compounds was carried out to identify the most potent heterocyclic substitution

The trans influence in unsymmetrical pincer palladacycles: an experimental and computational study

A library of unsymmetrical SCN pincer palladacycles, [ClPd{2-pyr-6-(RSCH2)C6H3}], R = Et, Pr, Ph, p-MePh, and p-MeOPh, pyr = pyridine, has been synthesized via C–H bond activation, and used, along with PCN and N’CN unsymmetrical pincer palladacycles previously synthesized by the authors, to determine the extent to which the trans influence is exhibited in unsymmetrical pincer palladacycles. The trans influence is quantified by analysis of structural changes in the X-ray crystal and density functional theory (DFT) optimized structures and a topological analysis of the electron density using quantum theory of atoms in molecules (QTAIM) to determine the strength of the Pd-donor atom interaction. It is found that the trans influence is controlled by the nature of the donor atom and although the substituents on the donor-ligand affect the Pd-donor atom interaction through the varied electronic and steric constraints, they do not influence the bonding of the ligand trans to it. The data indicate that the strength of the trans influence is P > S > N. Furthermore, the synthetic route to the family of SCN pincer palladacycles presented demonstrates the potential of late stage derivitization for the effective synthesis of ligands towards unsymmetrical pincer palladacycles

Elaboration of tetra-orthogonally-substituted aromatic scaffolds towards novel EGFR-kinase inhibitors

Nitration of three regioisomers of bromo-fluorobenzaldehyde proceeds regioselectively, notably with H2SO4/HNO3 at 0 °C. The thereby synthesized tetrasubstituted aromatics, endowed with orthogonal substituents, can be elaborated via Pd-catalysed coupling, reduction and reductive amination reactions. As a test-case, these compounds were converted into EGFR inhibitors related to Gefitinib, whose activity was rationalised by docking studies

Targeting cavity-creating p53 cancer mutations with small-molecule stabilizers: the Y220X paradigm

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket’s accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutant as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therap

Trial outcomes and information for clinical decision-making: a comparative study of opinions of health professionals

Background: Trials are robust sources of data for clinical practice, however, trial outcomes may not reflect what is important to communicate for decision making. The study compared clinicians’ views of outcomes to include in a core outcome set for colorectal cancer (CRC) surgery, with what clinicians considered important information for clinical practice (core information). Methods: Potential outcomes/information domains were identified through systematic literature reviews, reviews of hospital information leaflets and interviews with patients. These were organized into 6 categories, and used to design a questionnaire survey that asked surgeons and nurses from a sample of CRC centers to rate the importance of each domain as an outcome or as information on a 9-point Likert scale. Respondents were re-surveyed (round 2) following group feedback (Delphi methods). Comparisons were made by calculating the difference in mean scores between the outcomes and information domains, and paired t-tests were used to explore the difference between mean scores of the 6 outcome/information categories. Results: Data sources identified 1216 outcomes/information of CRC surgery that informed a 94 item questionnaire. First round questionnaires were returned from 63/81 (78%) of centers. Clinicians rated 76/94 (84%) domains of higher importance to measure in trials than to information to communicate to patients in Round 1. This was reduced to 24/47 (51%) in Round 2. The greatest difference was evident in domains about survival, which was rated much more highly as a trial outcome than important piece of information for decision-making (mean difference 2.3, 95% CI 1.9-2.8, p<0.0001). Specific complications and quality-of-life domains were rated similarly (mean difference 0.18, 95% CI -0.1-0.4, p=0.2 and 0.2, 95% CI -0.1-0.5, p=0.2 respectively). Conclusions: Whilst clinicians want to measure key outcomes in trials, they rate these as less important to communicate in decision-making with patients. This discrepancy needs to be explored and addressed to maximize the impact of trials on clinical practice

Extensive telomere erosion is consistent with localised clonal expansions in Barrett’s metaplasia

Barrett’s oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett’s oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett’s oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett’s metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett’s metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett’s metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett’s metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett’s metaplasia

Mobile Phone Radiation Induces Reactive Oxygen Species Production and DNA Damage in Human Spermatozoa In Vitro

Background: In recent times there has been some controversy over the impact of electromagnetic radiation on human health. The significance of mobile phone radiation on male reproduction is a key element of this debate since several studies have suggested a relationship between mobile phone use and semen quality. The potential mechanisms involved have not been established, however, human spermatozoa are known to be particularly vulnerable to oxidative stress by virtue of the abundant availability of substrates for free radical attack and the lack of cytoplasmic space to accommodate antioxidant enzymes. Moreover, the induction of oxidative stress in these cells not only perturbs their capacity for fertilization but also contributes to sperm DNA damage. The latter has, in turn, been linked with poor fertility, an increased incidence of miscarriage and morbidity in the offspring, including childhood cancer. In light of these associations, we have analyzed the influence of RF-EMR on the cell biology of human spermatozoa in vitro. Principal Findings: Purified human spermatozoa were exposed to radio-frequency electromagnetic radiation (RF-EMR) tuned to 1.8 GHz and covering a range of specific absorption rates (SAR) from 0.4 W/kg to 27.5 W/kg. In step with increasing SAR, motility and vitality were significantly reduced after RF-EMR exposure, while the mitochondrial generation of reactive oxygen species and DNA fragmentation were significantly elevated (P<0.001). Furthermore, we also observed highly significant relationships between SAR, the oxidative DNA damage bio-marker, 8-OH-dG, and DNA fragmentation after RF-EMRexposure. Conclusions: RF-EMR in both the power density and frequency range of mobile phones enhances mitochondrial reactive oxygen species generation by human spermatozoa, decreasing the motility and vitality of these cells while stimulating DNA base adduct formation and, ultimately DNA fragmentation. These findings have clear implications for the safety of extensive mobile phone use by males of reproductive age, potentially affecting both their fertility and the health and wellbeing of their offspring

The cost effectiveness of NHS physiotherapy support for occupational health (OH) services

Background: Musculoskeletal pain is detrimental to quality of life (QOL) and disruptive to activities of daily living. It also places a major economic burden on healthcare systems and wider society. In 2006, the Welsh Assembly Government (WAG) established a three tiered self-referral Occupational Health Physiotherapy Pilot Project (OHPPP) comprising: 1.) telephone advice and triage, 2.) face-to-face physiotherapy assessment and treatment if required, and 3.) workplace assessment and a return-to-work facilitation package as appropriate. This study aimed to evaluate the feasibility and cost-effectiveness of the pilot service. Methods: A pragmatic cohort study was undertaken, with all OHPPP service users between September 2008 and February 2009 being invited to participate. Participants were assessed on clinical status, yellow flags, sickness absence and work performance at baseline, after treatment and at 3 month follow up. Cost-effectiveness was evaluated from both top-down and bottom-up perspectives and cost per Quality Adjusted Life Year (cost/QALY) was calculated. The cost-effectiveness analysis assessed the increase in service cost that would be necessary before the cost-effectiveness of the service was compromised. Results A total of 515 patients completed questionnaires at baseline. Of these, 486 were referred for face to face assessment with a physiotherapist and were included in the analysis for the current study. 264 (54.3%) and 199 (40.9%) were retained at end of treatment and 3 month follow up respectively. An improvement was observed at follow up in all the clinical outcomes assessed, as well as a reduction in healthcare resource usage and sickness absence, and improvement in self-reported work performance. Multivariate regression indicated that baseline and current physical health were associated with work-related outcomes at follow up. The costs of the service were £194-£360 per service user depending on the method used, and the health gains contributed to a cost/QALY of £1386-£7760, which would represent value for money according to current UK thresholds. Sensitivity analyses demonstrated that the service would remain cost effective until the service costs were increased to 160% per user. Conclusions: This pragmatic evaluation of the OHPPP indicated that it was likely to be feasible in terms of service usage and could potentially be cost effective in terms of QALYs. Further, the study confirmed that improving physical health status for musculoskeletal pain patients is important in reducing problems with work capacity and related costs. This study suggests that this type of service could be potentially be useful in reducing the burden of pain and should be further investigated, ideally via randomised controlled trials assessing effectiveness and cost-effectiveness

The Effectiveness and Micro-costing Analysis of a Universal, School-Based, Social–Emotional Learning Programme in the UK: A Cluster-Randomised Controlled Trial

There are a growing number of school-based interventions designed to promote children’s social and emotional learning. One such intervention, PATHS (Promoting Alternative Thinking Strategies), was evaluated in a randomised controlled trial involving 5074 pupils aged 4–6 years at baseline in 56 primary schools across a large city in the UK. The programme was implemented for two academic years. The primary outcome measure was the teacher-rated Strengths and Difficulties Questionnaire (SDQ). A secondary measure was the PATHS Teacher Rating Scale (PTRS). Observations of child and teacher behaviours were undertaken in a third of intervention and control schools using the Teacher–Pupil Observation Tool (T-POT). Regarding fidelity, dose and adherence were measured via weekly logs completed by teachers, and a semi-structured questionnaire completed by PATHS coaches was used as a global measure of fidelity (capturing adherence, dose and quality). A cost-consequence analysis examined programme costs from a multi-agency public sector perspective. At 1 year post-baseline, there were no statistically significant differences between the programme and control groups on the SDQ subscales or the SDQ total difficulties and impact scores. There were statistically significant differences favouring the programme group for six out of 11 subscales on the secondary outcome measure (PTRS). At 2 years post-baseline, there were no statistically significant differences between the groups on either measure. Fidelity, according to the global measure, was relatively strong, and there was no relationship between fidelity and treatment effects. The average cost of PATHS was £12,666 per school or £139 per child. The study, which was fully powered and independent of the programme developer, shows no statistically significant effect of the programme on child behaviour or emotional well-being. Trial registration site and number: www.controlled-trials.com: ISRCTN 32534848

Immediate chest X-ray for patients at risk of lung cancer presenting in primary care: randomised controlled feasibility trial

Background: Achieving earlier stage diagnosis is one option for improving lung cancer outcomes in the United Kingdom. Patients with lung cancer typically present with symptoms to general practitioners several times before referral or investigation. Methods: We undertook a mixed methods feasibility individually randomised controlled trial (the ELCID trial) to assess the feasibility and inform the design of a definitive, fully powered, UK-wide, Phase III trial of lowering the threshold for urgent investigation of suspected lung cancer. Patients over 60, with a smoking history, presenting with new chest symptoms to primary care, were eligible to be randomised to intervention (urgent chest X-ray) or usual care. Results: The trial design and materials were acceptable to GPs and patients. We randomised 255 patients from 22 practices, although the proportion of eligible patients who participated was lower than expected. Survey responses (89%), and the fidelity of the intervention (82% patients X-rayed within 3 weeks) were good. There was slightly higher anxiety and depression in the control arm in participants aged >75. Three patients (1.2%) were diagnosed with lung cancer. Conclusions: We have demonstrated the feasibility of individually randomising patients at higher risk of lung cancer, to a trial offering urgent investigation or usual care