Publications

Degraders with dual activity against BRD4 and CBP/EP300 were designed. A structure-guided design approach was taken to assess and test potential exit vectors on the dual BRD4 and CBP/EP300 inhibitor, ISOX-DUAL. Candidate degrader panels revealed that VHL-recruiting moieties could mediate dose-responsive ubiquitination of BRD4. A panel of CRBN-recruiting thalidomide-based degraders was unable to induce ubiquitination or degradation of target proteins. High-resolution protein cocrystal structures revealed an unexpected interaction between the thalidomide moiety and Trp81 on the first bromodomain of BRD4. The inability to form a ternary complex provides a potential rationale for the lack of degrader activity with these compounds, some of which have remarkable affinities close to those of (+)-JQ1, as low as 65 nM in a biochemical assay, vs 1.5 μM for their POI ligand, ISOX-DUAL. Such a “degrader collapse” may represent an under-reported mechanism by which some putative degrader molecules are inactive with respect to target protein degradation.

NF-κB signalling is frequently dysregulated in human cancers making it an attractive therapeutic target. Despite concerted efforts to generate NF-κB inhibitors, direct pharmacological inhibition of the kinases mediating canonical NF-κB has failed due to on-target toxicities in normal tissues. So, alternative strategies, designed to target specific components of the NF-κB signalling machinery, have the potential to selectively inhibit tumour cells whilst reducing the toxicities associated with broad inhibition of NF-κB in non-malignant cells. Here we present evidence that a C8-linked pyrrolobenzodiazepine (PBD) containing proteolysis-targeting chimera (PROTAC) selectively degrades the NF-κB subunit, RelA/p65, in a proteasome-dependent manner. Our lead PROTAC (JP-163-16, 15d) showed cytotoxicity with mean LC50 values of 2.9 μM in MDA-MB-231 cells, 0.14 μM in MEC-1 cells and 0.23 μM in primary chronic lymphocytic leukaemia cells. In contrast, 15d was two-logs less toxic in primary B- and T-lymphocytes (mean LD50 19.1 μM and 36.4 μM, respectively). Importantly, the development of 15d, by conjugating the C8-linked PBD with a cereblon-targeting ligand using a five-carbon linker, abolished the ability of the C8-linked PBD to bind to DNA, whilst demonstrating cytotoxicity in cancer cells associated with the degradation of RelA/p65. Mechanistically, 15d displayed PROTAC credentials through the selective degradation of NF-κB RelA/p65 in a proteasome-dependent manner and showed a five-fold reduction in potency in the cereblon deficient, lenalidomide resistant, myeloma cell line, RPMI-8226. To our knowledge, this work describes the first PROTAC capable of selective degradation of a single NF-κB subunit and highlights the therapeutic potential of our strategy for the treatment of RelA/p65-dependent tumours.

No Abstract Found.

Upon infection, human papillomavirus (HPV) manipulates host cell gene expression to create an environment that is supportive of a productive and persistent infection. The virus-induced changes to the host cell’s transcriptome are thought to contribute to carcinogenesis. Here, we show by RNA-sequencing that oncogenic HPV18 episome replication in primary human foreskin keratinocytes (HFKs) drives host transcriptional changes that are consistent between multiple HFK donors. We have previously shown that HPV18 recruits the host protein CTCF to viral episomes to control the differentiation-dependent viral transcriptional programme. Since CTCF is an important regulator of host cell transcription via coordination of epigenetic boundaries and long-range chromosomal interactions, we hypothesised that HPV18 may also manipulate CTCF to contribute to host transcription reprogramming. Analysis of CTCF binding in the host cell genome by ChIP-Seq revealed that while the total number of CTCF binding sites is not altered by the virus, there are a sub-set of CTCF binding sites that are either enriched or depleted of CTCF. Many of these altered sites are clustered within regulatory elements of differentially expressed genes, including the tumour suppressor gene cell adhesion molecule 1 (CADM1), which supresses epithelial cell growth and invasion. We show that HPV18 establishment results in reduced CTCF binding at the CADM1 promoter and upstream enhancer. Loss of CTCF binding is coincident with epigenetic repression of CADM1, in the absence of CpG hypermethylation, while adjacent genes including the transcriptional regulator ZBTB16 are activated. These data indicate that the CADM1 locus is subject to topological rearrangement following HPV18 establishment. We tested this hypothesis using 4C-Seq (circular chromosome confirmation capture-sequencing) and show that HPV18 establishment causes a loss of long-range chromosomal interactions between the CADM1 transcriptional start site and the upstream transcriptional enhancer. These data show that HPV18 manipulates host cell promoter-enhancer interactions to drive transcriptional reprogramming that may contribute to HPV-induced disease progression.

Maintaining genome integrity is an essential and challenging process. RAD51 recombinase, the central component of several crucial processes in repairing DNA and protecting genome integrity, forms filaments on DNA, which are tightly regulated. One of these RAD51 regulators is FIGNL1 (fidgetin-like 1), which prevents RAD51 genotoxic chromatin association in normal cells and persistent RAD51 foci upon DNA damage. The cryogenic electron microscopy–imaged structure of FIGNL1 in complex with RAD51 reveals that FIGNL1 forms a nonplanar hexamer and encloses RAD51 N terminus in the FIGNL1 hexamer pore. Mutations in pore loop or catalytic residues of FIGNL1 render it defective in filament disassembly and are lethal in mouse embryonic stem cells. Our study reveals a distinct mechanism for removing RAD51 from bound substrates and provides the molecular basis for FIGNL1 in maintaining genome stability.

The P2X purinergic receptor 7 (P2X7) has an essential role in inflammation, innate immunity, tumor progression, neurodegenerative diseases, and several other diseases, leading subsequently to the development of P2X7 modulators. AZ11645373 is a frequently studied P2X7 antagonist tool compound but always used as a racemic mixture. Racemic AZ11645373 can be separated into its respective enantiomers by chiral chromatography, albeit in small batches, and these were stereochemically intact over two years later, by chiral high-performance liquid chromatography (HPLC) analysis. On a higher scale, significant decomposition is observed during purification. One of the enantiomers was crystallized as a palladium complex, and its (R)-configuration was determined by single-crystal X-ray diffraction, further confirmed, in solution, by vibrational circular dichroism. Biological studies demonstrated that both (S)- and (R)-forms were able to fully inhibit human P2X7, but (R)-AZ11645373 was more potent, with an IC50 of 32.9 nM. Contrary to its effect on human P2X7, (S)-AZ11645373 was ineffective on mouse P2X7, while the (R)-AZ11645373 enantiomer was a full antagonist. These results demonstrated that the antagonistic effects of racemic AZ11645373 are mainly due to its (R)-enantiomer. Site-directed mutagenesis and molecular dynamics simulations indicated that the (R)-enantiomer may form specific interactions with Phe95 and the antagonists bound to other P2X7 monomers. Phe95 is situated in the allosteric binding site at the edge of the upper vestibule and appears to be the pivotal molecular gateway between AZ11645373 allosteric binding and locking of the closed state of the P2X7 channel. All together, these structure-function relationships should be helpful for drug design of P2X7 modulators.

Introduction: Physiological age-related bone loss is common, with 50% of women aged ≥80 having osteoporosis. Bone loss is exacerbated in women receiving aromatase inhibitors (AIs) for early breast cancer (EBC), increasing fracture risk. This study explored the management of bone-health in older women (≥70 years) with EBC and factors influencing clinical decision-making. Materials and Methods: This was a sub-study of a larger United Kingdom multicentre observational study into practice variation and outcomes in older women (≥70) with EBC (Age Gap study). Participants were aged ≥70 years with EBC; data were collected on health status, treatments, and outcomes. This sub-study focused on patients recruited at five hospitals, where more detailed data on bone health and management were collected for women with ER + ve (oestrogen receptor positive) cancers who received adjuvant or primary endocrine therapy treatment. We aimed to determine factors influencing treatment selection and outcomes in this age group. Results: The main Age Gap study recruited between 2013 and 2018. In this sub-study, 565 patients had ER + ve cancers, of whom 529 (93.6%) received AIs and 26 (4.6%) tamoxifen. The median age of participants was 77 years (70–98 years). A baseline dual energy x-ray absorptiometry (DEXA) scan was performed in only 354/529 (67%) of the AI group. Bisphosphonates were prescribed for 226/529 (43%). Baseline DEXA scans were more likely to be requested if patients were fit for surgery and were < 80 years old. Of those scanned (n = 354), 148 (42%) were osteopenic and 64 (18%) osteoporotic. Bisphosphonate prescription was associated with younger age (<80 years old) (p = 0.02). From recruitment to 2022, fractures were diagnosed in 23% of participants (122/529), of whom only 38% (46/122) had received prior bisphosphonates. Frailty or prefrailty (Rockwood scale) were present in 94% (431/461), but there was no correlation between frailty and baseline hip (r2 = 0.0098) or spine (r2 = 0.00007) T-scores. Rates of DEXA scanning varied between centres from 36% to 76% (p < 0.001) for unknown reasons. Discussion: Age and general health influenced bone-health management decision-making, but there was considerable variation between centres, highlighting the need for standardised bone-health care for older women with EBC.

Wnt/β-catenin signalling is important for normal hematopoietic stem/progenitor cell (HSPC) biology and heavily implicated in acute and chronic myeloid leukaemia (AML and CML). The central mediator β-catenin is an attractive therapeutic target in myeloid neoplasms however its targeting has been hampered by a poor characterisation of its molecular interactions in haematopoietic cells, which will differ from its network in solid tissues. Our previous β-catenin interactome study identified the significant enrichment of RNA-binding proteins (RBP) implying post-transcriptional roles for β-catenin in myeloid cells. To identify β-catenin interacting mRNAs we performed β-catenin RNA-immunoprecipitation coupled to RNA-sequencing (RIP-seq) and identified significantly enriched Wnt signalling pathway transcripts. Using β-catenin cross-linking immunoprecipitation (CLIP) we demonstrated a limited capacity for β-catenin to bind RNA directly, implying dependence on other RBPs. β-Catenin was found to interact with Musashi-2 (MSI2) in both myeloid cell lines and primary AML patient samples, where expression was significantly correlated. MSI2 knockdown reduced Wnt signalling output (TCF/LEF activity), through suppression of LEF-1 expression and nuclear localisation. Through both RIP and CLIP we demonstrate MSI2 binds LEF1 mRNA in a partly β-catenin dependent fashion, and may impact the post-transcriptional control of LEF-1 expression. Finally, we show that MSI2-mediated expansion of human HSPCs could be partly driven through LEF1 regulation. This is the first study to experimentally demonstrate functional crosstalk between MSI2 and Wnt signalling in human cells, and indicates potential novel post-transcriptional roles for β-catenin in a haematological context.

The Wnt/β-catenin pathway is an evolutionarily conserved signal transduction cascade with critical regulatory roles in cellular proliferation, cell fate determination and tissue homeostasis. Through the regulation of multiple human stem cell systems, canonical Wnt signalling is not only a major contributor to normal development, but also heavily implicated in a multitude of human diseases, including cancer. The central mediator of the pathway β-catenin, first identified as Armadillo (ARM) in Drosophila, has well-defined roles in cell adhesion and transcription within the pathway. However, accumulating evidence suggests β-catenin functionality is more complex than initially anticipated with reported roles beyond those historically characterised, including the regulation of RNA and RNA-binding proteins (RBP). This review will summarise the current understanding around β-catenin as a post-transcriptional regulator in normal and malignant development, drawing particular attention to cell types not traditionally used to characterise Wnt signalling but uniquely placed to reveal novel β-catenin function.

No Abstract Found

Objectives: Androgen deprivation therapy (ADT) forms the mainstay of treatment for advanced prostate cancer. Traditionally administered as a luteinising hormone-releasing hormone (LHRH) agonist depot injection, newer options for ADT include transdermal oestradiol patches (tE2) or oral LHRH antagonists. This study aimed to identify whether this is an important choice for men, which treatment men would choose if offered either LHRH agonist injections, tE2 patches or oral LHRH antagonists as ADT, and to explore the factors influencing this decision. Subjects, Patients, and Methods: Five focus groups were conducted. A total of 24 men from around the UK participated in discussions, of whom 10 had never had prostate cancer and 14 had early prostate cancer but had not received ADT. Focus groups were co-facilitated with patient and public involvement representatives. Transcripts were analysed using a critical realist thematic analysis approach. Results: Participants reported that having a choice of ADT is important and being involved in making treatment decisions can help men maintain an element of control. Most men expressed a preference to avoid ADT with LHRH agonist injections; 14 of the 24 men reported they would choose an oral LHRH antagonist, eight reported they would choose tE2 patches, and two that they would choose LHRH agonist injections. Participants reported a large number of factors that influenced their treatment choice that were grouped into: (i) side-effects of treatment, (ii) logistical aspects, and (iii) advice from trusted others. Personal preferences between different types of ADT were based on factors that participants identified as important to them, this prioritisation of factors was influenced by participants’ past experiences, current health beliefs, and future expectations. Conclusions: Men wish to have choice of ADT, and many would not choose LHRH agonists injections if there were other ADT options available. This should be considered, as reimbursement committees and guideline-makers consider the role of alternative ADT strategies.

Copper catalyzed aldehyde-alkyne-amine (A3) couplings lead to multifunctional, racemic, propargylic amines, many on a multigram scale. As part of an industrial collaboration, a selection of linkers was purified by chiral HPLC to afford single enantiomers, the absolute configuration of which was determined by vibrational circular dichroism (vCD). To show medicinal chemistry applications, selected linkers were further derivatized into potential cellular probes and (+)-JQ1 containing PROTACs (proteolysis targeting chimeras), which degraded their target protein BRD4.

Fatigue may affect the decision to deploy effort (cost) for a given rewarded outcome (benefit). However, it remains unclear whether these fatigue-associated changes can be attributed to simply feeling fatigued. To investigate this question, twenty-two healthy males made a series of choices between two rewarded options: a fixed, no effort option, where no physical effort was required to obtain a set, low reward vs. a variable, effortful option, in which both the physical effort requirements (i.e. different durations of a sustained contraction performed in the lower limbs) and rewards (i.e. greater monetary incentives) was varied. Effort-based choices were made under two conditions: (1) a rested (control) state, (2) a pre-induced state of perceived fatigue, evoked through physical exertion in the upper-limbs, thus controlling for the physical manifestation of fatigue (i.e. decline in muscular force production in the lower limbs) on the decision process. Though prior physical exertion increased the perception of fatigue, participants choices did not significantly differ between control and fatigue conditions. Across both conditions, participants demonstrated an anticipated aversion to effort, with greater effort requirements reducing the decision to engage in actions associated with higher rewards. However, in the fatigue state only, decision time was prolonged and self-reported confidence in individuals’ ability to perform high effort actions was reduced. The findings suggest that a perceived state of fatigue does not necessarily alter cost/benefit comparisons within effort-based decisions, but may introduce greater uncertainty within choice and reduce self-confidence. These findings evidence altered evaluative processes during decision making under conditions of fatigue.

This review comprehensively analyzes key parameters that gov-ern the use of luminescent benzoselenadiazole (BSD) derivatives.We examine the main factors affecting their photophysicalproperties, including structural variations, heavy atom effectsand environmental conditions, and discuss their potentialapplications in bioimaging technology. Whenever possible, theproperties were compared to those of 2,1,3-benzoxadiazole and2,1,3-benzothiadiazole analogues. This review also highlightsemerging trends and future directions in the research and devel-opment of BSD compounds, underscoring their significance andpotential impact in advancing phototechnology and relatedfields. We also aim to offer valuable insight into the versatilenature of BSD derivatives and their expanding role in scientificand technological innovations.