The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report ...cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) ...sub-family are 2-oxoglutarate (2-OG) and Fe2+-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation.
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•KDOAM-25 is a selective inhibitor of KDM5A-D, the H3K4me3 lysine demethylases•KDOAM-25 inhibits the demethylation of H3K4me3 in an immunofluorescence assay•Increased KDM5B expression correlates with shorter survival in multiple myeloma•KDOAM-25-treated cells show an increase in H3K4me3 at transcription start sites
Tumber et al. report the discovery and characterization of KDOAM-25, a potent and selective inhibitor of the KDM5 H3K4me3 lysine demethylases. KDOAM-25 inhibits the cellular demethylation of H3K4me3 at transcription start sites and the proliferation of MM1S multiple myeloma cells.
We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. ...1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household contact were at greatest risk (adjusted odds ratio aOR 4.82 95%CI 3.45-6.72). Higher rates of Covid-19 were seen in staff working in Covid-19-facing areas (22.6% vs. 8.6% elsewhere) (aOR 2.47 1.99-3.08). Controlling for Covid-19-facing status, risks were heterogenous across the hospital, with higher rates in acute medicine (1.52 1.07-2.16) and sporadic outbreaks in areas with few or no Covid-19 patients. Covid-19 intensive care unit staff were relatively protected (0.44 0.28-0.69), likely by a bundle of PPE-related measures. Positive results were more likely in Black (1.66 1.25-2.21) and Asian (1.51 1.28-1.77) staff, independent of role or working location, and in porters and cleaners (2.06 1.34-3.15).
We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent ...kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.
We investigated determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike IgG responses in healthcare workers (HCWs) following one or two doses of Pfizer–BioNTech or ...Oxford–AstraZeneca vaccines.
HCWs participating in regular SARS-CoV-2 PCR and antibody testing were invited for serological testing prior to first and second vaccination, and 4 weeks post-vaccination if receiving a 12-week dosing interval. Quantitative post-vaccination anti-spike antibody responses were measured using the Abbott SARS-CoV-2 IgG II Quant assay (detection threshold: ≥50 AU/mL). We used multivariable logistic regression to identify predictors of seropositivity and generalized additive models to track antibody responses over time.
3570/3610 HCWs (98.9%) were seropositive >14 days post first vaccination and prior to second vaccination: 2706/2720 (99.5%) were seropositive after the Pfizer–BioNTech and 864/890 (97.1%) following the Oxford–AstraZeneca vaccines. Previously infected and younger HCWs were more likely to test seropositive post first vaccination, with no evidence of differences by sex or ethnicity. All 470 HCWs tested >14 days after the second vaccination were seropositive. Quantitative antibody responses were higher after previous infection: median (IQR) >21 days post first Pfizer–BioNTech 14 604 (7644–22 291) AU/mL versus 1028 (564–1985) AU/mL without prior infection (p < 0.001). Oxford–AstraZeneca vaccine recipients had lower readings post first dose than Pfizer–BioNTech recipients, with and without previous infection, 10 095 (5354–17 096) and 435 (203–962) AU/mL respectively (both p < 0.001 versus Pfizer–BioNTech). Antibody responses >21 days post second Pfizer vaccination in those not previously infected, 10 058 (6408–15 582) AU/mL, were similar to those after prior infection followed by one vaccine dose.
SARS-CoV-2 vaccination leads to detectable anti-spike antibodies in nearly all adult HCWs. Whether differences in response impact vaccine efficacy needs further study.
In clear cell renal cell carcinoma, 80% of cases have biallelic inactivation of the VHL gene, leading to constitutive activation of both HIF1α and HIF2α. As HIF2α is the driver of the disease ...promoting tumour growth and metastasis, drugs targeting HIF2α have been developed. However, resistance is common, therefore new therapies are needed.
We assessed the effect of the HIF2α antagonist PT2385 in several steps of tumour development and performed RNAseq to identify genes differentially expressed upon treatment. A drug screening was used to identify drugs with antiproliferative effects on VHL-mutated HIF2α-expressing cells and could increase effectiveness of PT2385.
PT2385 did not reduce cell proliferation or clonogenicity but, in contrast to the genetic silencing of HIF2α, it reduced in vitro cell invasion. Many HIF-inducible genes were down-regulated upon PT2385 treatment, whereas some genes involved in cell migration or extracellular matrix were up-regulated. HIF2α was associated with resistance to statins, addition to PT2385 did not increase the sensitivity.
this study shows key differences between inhibiting a target versus knockdown, which are potentially targetable.
Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell‐permeable inhibitors or suitable tool compounds for ...these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure–activity relationships, leading to the development of a small molecule with around 75‐fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.
Small but mighty: A new class of inhibitor was discovered that is highly potent towards the histone lysine demethylases (KDMs) KDM2A/7A. A modular synthetic approach was used to explore chemical space and accelerate the investigation of key structure–activity relationships, which led to the development of a small molecule with more than 75‐fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.
Cyclins are central engines of cell cycle progression in conjunction with cyclin‐dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner ...with a CDK, but instead forms via its F‐box domain an SCF (Skp1‐Cul1‐F‐box)‐type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F‐depleted cells. We find that SCF‐cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.
Synopsis
A kinase inhibitor screen for synthetic lethality in cells lacking the ubiquitin ligase adaptor Cyclin F leads to the identification of E2F1 as new SCF‐Cyclin F substrate, whose unrestricted activity promotes catastrophic replication stress upon checkpoint suppression with Chk1 inhibitors.
A screen to identify vulnerabilities of cells lacking cyclin F identifies Chk1 inhibitors as primary hits.
Cyclin F loss predisposes cells to the toxic effect of Chk1i by inducing DNA replication catastrophe.
Aberrant accumulation of E2F1 upon cyclin F loss promotes DNA damage upon Chk1 inhibition.
Lack of cyclin F prevents degradation of E2F1 in G2/M and upon Chk1 inhibitors.
A non‐degradable E2F1 lacking a CY motif induces cell death and DNA replication stress upon Chk1 inhibition.
A kinase inhibitor screen reveals that Chk1‐dependent checkpoint responses are required to prevent catastrophic replication stress caused by unrestricted E2F1 activity in cells lacking Cyclin F.
Thresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. In this setting the sensitivity and specificity of the best ...performing assays can both exceed 98%. However, antibody assay performance following mild infection is less clear.
We assessed quantitative IgG responses in a cohort of healthcare workers in Oxford, UK, with a high pre-test probability of Covid-19, in particular the 991/11,475(8.6%) who reported loss of smell/taste. We use anosmia/ageusia and other risk factors as probes for Covid-19 infection potentially undiagnosed by immunoassays by investigating their relationship with antibody readings either side of assay thresholds.
The proportion of healthcare workers reporting anosmia/ageusia increased at antibody readings below diagnostic thresholds using an in-house ELISA (n = 9324) and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA; n = 11,324): 426/906 (47%) reported anosmia/ageusia with a positive ELISA, 59/449 (13.1%) with high-negative and 326/7969 (4.1%) with low-negative readings. Similarly, by CMIA, 518/1093 (47.4%) with a positive result reported anosmia/ageusia, 106/686 (15.5%) with a high-negative and 358/9563 (3.7%) with a low-negative result. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays in mild infection is lower than previously reported: Oxford ELISA 89.8% (95%CI 86.6-92.8%) and Abbott CMIA 79.3% (75.9-82.7%).
Following mild SARS-CoV-2 infection 10-30% of individuals may have negative immunoassay results. While lowered diagnostic thresholds may result in unacceptable specificity, our findings have implications for epidemiological analyses and result interpretation in individuals with a high pre-test probability. Samples from mild PCR-confirmed infections should be included in SARS-CoV-2 immunoassay evaluations.
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