Lessons from LIMK1 enzymology and their impact on inhibitor design

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique ‘rock-and-poke’ mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding

The uses and functions of ageing celebrity war reporters

This article starts from the premise that recognition of professional authority and celebrity status depends on the embodiment and performance of field-specific dispositional practices: there’s no such thing as a natural, though we often talk about journalistic instinct as something someone simply has or doesn’t have. Next, we have little control over how we are perceived by peers and publics, and what we think are active positioning or subjectifying practices are in fact, after Bourdieu, revelations of already-determined delegation. The upshot is that two journalists can arrive at diametrically opposed judgements on the basis of observation of the same actions of a colleague, and as individuals we are blithely hypocritical in forming (or reciting) evaluations of the professional identity of celebrities. Nowhere is this starker than in the discourse of age-appropriate behaviour, which this paper addresses using the examples of ‘star’ war reporters John Simpson, Kate Adie and Martin Bell. A certain rough-around-the-edges irreverence is central to dispositional authenticity amongst war correspondents, and for ageing hacks this incorporates gendered attitudes to sex and alcohol as well as indifference to protocol. And yet perceived age-inappropriate sexual behaviour is also used to undermine professional integrity, and the paper ends by outlining the phenomenological context that makes possible this effortless switching between amoral and moralising recognition by peers and audiences alike

Structural basis for binding of the renal carcinoma target hypoxia-inducible factor 2α to prolyl hydroxylase domain 2

The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment

Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1–3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1–3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design

Selective targeting of human TET1 by cyclic peptide inhibitors: Insights from biochemical profiling

Ten-Eleven Translocation (TET) enzymes are Fe(II)/2OG-dependent oxygenases that play important roles in epigenetic regulation, but selective inhibition of the TETs is an unmet challenge. We describe the profiling of previously identified TET1-binding macrocyclic peptides. TiP1 is established as a potent TET1 inhibitor (IC50 = 0.26 \ub5M) with excellent selectivity over other TETs and 2OG oxygenases. TiP1 alanine scanning reveals the critical roles of Trp10 and Glu11 residues for inhibition of TET isoenzymes. The results highlight the utility of the RaPID method to identify potent enzyme inhibitors with selectivity over closely related paralogues. The structure–activity relationship data generated herein may find utility in the development of chemical probes for the TETs

Journalism Studies

This entry traces the history of journalism studies and asks whether journalism studies are a discipline or field or research method. Different interests involved in journalism studies – journalists, journalism educators and journalism scholars - make it difficult to find a single vision of what it entails. As a new field it requires its own methodologies even though these may be borrowed from other disciplines. It also requires its own body of literature. The origins of journalism studies are somewhat imprecise but we can identify five phases of evolution: normative, empirical turn, sociological turn, global-comparative turn, and digital turn. Journalism studies also encompass the education of journalists. Many journalism scholars now reside in journalism departments side by side with their practitioner colleagues. Historically the study and practice of journalism was entwined over the debate of whether the occupation of journalism should be regarded as a craft or a profession and indeed its place in the academy

Cyclic β^2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease

Due to their constrained conformations, cyclic β2,3-amino acids (cβAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cβAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cβAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating three kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β1), (1S,2S)-2-aminocyclohexane carboxylic acid (β2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one β2 and two β1, exhibited potent inhibitory activities with IC50 values of 40 nM and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 h and >168 h, respectively. Notably, BM3A and BM7A, wherein the cβAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties

Chinese-style pragmatic objectivity in war reporting

China plays an increasing role in the wars and conflicts around the world with its expanding political and economic interests overseas, and its diplomatic role in international affairs. More and more Chinese journalists go to the frontlines overseas to cover distant conflicts for domestic audiences. Based on semi-structured in-depth interviews with sixteen Chinese correspondents who have covered conflicts outside China, this study examines Chinese journalists’ perceptions and reflections on objectivity in the war zones. The author adopts a term of Chinese-style pragmatic objectivity to mean that objectivity is a convenient approach for Chinese journalists to do war journalism in the field. At the level of objectivity-as-a- value, objectivity is defined as a pragmatic value and a practical ritual for Chinese journalists to do news within the scope they can reach, to protect themselves from criticisms, and to justify their version of the truth. It promotes allegiance and patriotism. At the level of objectivity-as-a-practice, objectivity in war coverage is compromised by China’s foreign policies, military constraints, the press’s political orientations and editorial polices, and journalists’ personal experiences and values. Chinese journalists use Chinese-style objectivity to negotiate their roles in the power struggle with the state, foreign militaries, the newsroom and journalists

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6

The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia

Comparative Natural History of Visual Function From Patients With Biallelic Variants in BBS1 and BBS10.

The purpose of this study was to compare the natural history of visual function change in cohorts of patients affected with retinal degeneration due to biallelic variants in Bardet-Biedl syndrome genes: BBS1 and BBS10. Patients were recruited from nine academic centers from six countries (Belgium, Canada, France, New Zealand, Switzerland, and the United States). Inclusion criteria were: (1) female or male patients with a clinical diagnosis of retinal dystrophy, (2) biallelic disease-causing variants in BBS1 or BBS10, and (3) measures of visual function for at least one visit. Retrospective data collected included genotypes, age, onset of symptoms, and best corrected visual acuity (VA). When possible, data on refractive error, fundus images and autofluorescence (FAF), optical coherence tomography (OCT), Goldmann kinetic perimetry (VF), electroretinography (ERG), and the systemic phenotype were collected. Sixty-seven individuals had variants in BBS1 (n = 38; 20 female patients and 18 male patients); or BBS10 (n = 29; 14 female patients and 15 male patients). Missense variants were the most common type of variants for patients with BBS1, whereas frameshift variants were most common for BBS10. When ERGs were recordable, rod-cone dystrophy (RCD) was observed in 82% (23/28) of patients with BBS1 and 73% (8/11) of patients with BBS10; cone-rod dystrophy (CORD) was seen in 18% of patients with BBS1 only, and cone dystrophy (COD) was only seen in 3 patients with BBS10 (27%). ERGs were nondetectable earlier in patients with BBS10 than in patients with BBS1. Similarly, VA and VF declined more rapidly in patients with BBS10 compared to patients with BBS1. Retinal degeneration appears earlier and is more severe in BBS10 cases as compared to those with BBS1 variants. The course of change of visual function appears to relate to genetic subtypes of BBS