Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.     

The Emerging Role of Social Media in Urology

Social media have become so integrated into modern communications as to be universal in our personal and, increasingly, professional lives. Recent examples of social media uptake in urology, and the emergence of data to quantify it, reveal the expansion of conventional communication routes beyond the in-person forum. In every domain of urologic practice, from patient interaction through research to continuing professional development, the move online has unlocked another layer of conversation, dissemination, and, indeed, caveats. Social media have a democratizing effect, placing patients, trainees, practitioners, and thought leaders in the same arena and on equal footing. If uptake of social media in medicine even remotely parallels its rise to ubiquity in other areas, it will only expand and evolve in the coming years. For these reasons, this article presents an overview of the most recent data on the impact and potential complications of social media usage in the urologic community.Key words: Social media, Urologic practice, Education, Continuing professional development, Research publication and impactInterprofessional communication and continuing professional development of and by physicians have typically involved face-to face and in-person interaction, in meeting rooms or lecture halls. Dissemination of research findings has occurred through publication in print (and, more recently, online) journals, sifting through the editorial process and with limited potential for interaction; replies and queries regarding a study typically appear months after its initial publication. Citation in subsequent research papers is the foundation of measurement of a journal’s impact.Social media may be broadly defined as those Internet-based sites and services that are primarily conversational in nature. Users post information or opinion, and other users, either through curated access lists or public profiles, are able to comment on or share the original post. The standard bearers in the last several years have been services such as Facebook, boasting over 1 billion active users, and Twitter, with well over 200 million active users.^1,2 Despite having, in large part, existed for less than a decade, these and many other services have become ubiquitous in personal and public life. In professional settings, however, uptake has been relatively guarded.^3,4 Recent examples of social media uptake in urology, and the emergence of data to support it, reveal the expansion of conventional communication routes beyond the in-person forum. Journal clubs have emerged with international participation, and the use of Twitter at medical meetings has added a conversational element and dissemination potential that had not previously existed. Publication of research and measurement of its impact are also in evolution, with potential for rapid propagation of research findings and impact metrics at the article level. Concerns have emerged in step with these innovations, such as consternation about patient (and physician) privacy and boundaries, as well as risks of posting words or images that may be, or are perceived to be, unprofessional.     

The JAK3 inhibitor WHI-P154 prevents PDGF-evoked process outgrowth in human neural precursor cells

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a novel mechanism for treating a variety of CNS disorders. The aim of this study was to investigate pathways controlling neurite outgrowth in human neural precursor cells, in particular in response to platelet-derived growth factor (PDGF). PDGF-AA, -AB and -BB were found to initiate calcium signalling and produce robust increases in neurite outgrowth. PDGF-induced outgrowth of Tuj1-positive precursors was abolished by the addition of EGTA, suggesting that calcium entry is a critical part of the signalling pathway. Wortmannin and PD098059 failed to inhibit PDGF-induced outgrowth. Clostridium Toxin B increased the amount of PDGF-induced neurite branching but had no effect on basal levels. In contrast, WHI-P154, an inhibitor of Janus protein tyrosine kinase (JAK3), Hck and Syk, prevented PDGF-induced neurite outgrowth. PDGF activates multiple signalling pathways with considerable potential for cross-talk. This study has highlighted the complexity of the pathways leading to neurite outgrowth in human neural precursors, and provided initial evidence to suggest that calcium entry is critical in producing the morphological changes observed.     

Assessment of the Target Engagement and d-Serine Biomarker Profiles of the d-Amino Acid Oxidase Inhibitors Sodium Benzoate and PGM030756

Neurochemical Research - Irregular N-methyl-d-aspartate receptor (NMDAR) function is one of the main hypotheses employed to facilitate understanding of the underlying disease state of...     

Lead discovery for microsomal prostaglandin E synthase using a combination of high-throughput fluorescent-based assays and RapidFire mass spectrometry

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.     

Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire mass spectrometry

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.