Diversity-oriented synthesis encoded by deoxyoligonucleotides.

Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Australian medical students' and junior doctors' perceptions of gender discrepancies in obstetrics and gynaecology.

BACKGROUND: There is currently a gender imbalance 85:15 female/male in the intake into specialist training for the Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG). AIMS: To determine the views and perceptions of Australian medical students, and junior doctors in the first five years of practice, toward obstetrics and gynaecology (O&G) as a career, including whether there are any perceived barriers to the pursuit of such a career. MATERIALS AND METHODS: A semi-structured questionnaire was developed with members of the RANZCOG Gender Equity and Diversity Working Group There were two separate studies: the first involved telephone interviews of medical students across three campuses of a medical school in North Queensland. The second study surveyed junior doctors in Queensland who are members of the Australian Medical Association. Responses were analysed and compared using quantitative and qualitative methods. RESULTS: Both studies found that experiences with O&G as a medical student influenced the decision to pursue O&G as a career. Exclusion from clinical scenarios and difficulty establishing good relationship with midwives within busy birthing suites were some reasons deterring male students from O&G. In addition, students felt poorly informed about the specialty in their preclinical years, affecting their early decisions in choice of specialty. Post-rotation, more female than male students reported positive experiences and were considering O&G as a career. CONCLUSIONS: Both groups see medical student experience as critical in attitudes toward the specialty as a possible career. This experience plays a significant role in encouraging female students toward a career in O&G and discouraging male students. More exposure to the specialty in the preclinical years, and attention to improving clinical rotations for all students, is required.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

The single-cell eQTLGen consortium.

In recent years, functional genomics approaches combining genetic information with bulk RNA-sequencing data have identified the downstream expression effects of disease-associated genetic risk factors through so-called expression quantitative trait locus (eQTL) analysis. Single-cell RNA-sequencing creates enormous opportunities for mapping eQTLs across different cell types and in dynamic processes, many of which are obscured when using bulk methods. Rapid increase in throughput and reduction in cost per cell now allow this technology to be applied to large-scale population genetics studies. To fully leverage these emerging data resources, we have founded the single-cell eQTLGen consortium (sc-eQTLGen), aimed at pinpointing the cellular contexts in which disease-causing genetic variants affect gene expression. Here, we outline the goals, approach and potential utility of the sc-eQTLGen consortium. We also provide a set of study design considerations for future single-cell eQTL studies.

Small-Molecule and CRISPR Screening Converge to Reveal Receptor Tyrosine Kinase Dependencies in Pediatric Rhabdoid Tumors.

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.

Medical students and midwives - How do they view each other?

Medical students from James Cook University who had completed their rotation in obstetrics, and midwives working in Cairns Hospital who had undertaken supervision of medical students in the birth suite, were invited to complete anonymous questionnaires on their views of their respective roles in the birth suite. Several issues were identified including increased medical and midwifery student numbers, and lack of communication between midwives and medical students. Increased cooperation and communication between medical and midwifery education providers is urgently needed to improve both student groups' learning experiences.

Retrograde type A dissection following hybrid supra-aortic endovascular surgery in high-risk patients unfit for conventional open repair.

BACKGROUND: Hybrid procedures with combined open extra-anatomical supra-aortic bypasses and endovascular surgery are less invasive for patients with complex aortic arch pathology. The aim of this paper is to report patients who developed retrograde type A aortic dissection following initially successful hybrid endovascular treatment. METHODS: Retrospective review of prospectively collected computerized departmental database. All patients with supra-aortic hybrid endovascular surgery and post-procedure retrograde type A dissection were identified. Patient demographics, comorbid conditions, perioperative parameters, procedural details and post-operative complications were collected. RESULTS: From May 2005 to July 2014, 163 patients underwent thoracic aortic endovascular procedures at our institution. From the 46 patients who had supra-aortic hybrid endovascular repair, six patients (6/46, 13% of all supra-aortic hybrid cases, 3 males) developed retrograde type A aortic dissection. All were elective cases, with 3 chronic dissecting aneurysms and 3 atherosclerotic aneurysms. All had one-stage hybrid procedures: 2 patients had carotid-carotid bypass grafts, one had carotid-carotid-left subclavian bypass graft, and 3 had bypass grafts from ascending aorta to innominate artery and left carotid artery. Five patients had Cook Zenith thoracic stent-grafts (Cook Medical, Bloomington, IN, USA), and one had Medtronic Valiant stent-grafts (Medtronic Vascular Inc, Santa Rosa, CA, USA). The retrograde type A dissection occurred with sudden symptoms at day 5, 6, 10, 20, 105 and 128, respectively. There were 3 immediate fatalities and 2 patients treated conservatively deemed unfit for reintervention (one died of pneumonia at 9 months, and one remained alive at 7 months post-complication). One patient underwent successful emergency open surgery and survived. CONCLUSIONS: Supra-aortic hybrid procedures in treating aortic arch pathology may be at risk of developing retrograde type A dissection. This post-operative complication inevitably have poor outcome, even with early diagnosis and prompt treatment.

GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.

Review of peripartum hysterectomy rates at a tertiary Australian hospital.

BACKGROUND: Peripartum hysterectomy is commonly performed for catastrophic postpartum haemorrhage uncontrolled by conservative medical and surgical therapies. Currently, information about the incidence and indications for peripartum hysterectomy are not well defined in Australia. AIMS: Evaluate the incidence and indications of peripartum hysterectomy in the Royal Brisbane and Women's Hospital (RBWH) between 2000 and 2014. MATERIALS AND METHODS: A 15-year retrospective cohort study of peripartum hysterectomies at RBWH was conducted. The incidence of this event was calculated. Risk factors for abnormal placentation were explored using univariate analyses. Statistical significance was declared at α < 0.05. RESULTS: A total of 83 cases of peripartum hysterectomy were reviewed. The incidence of peripartum hysterectomy was 0.60 per 1000 births after discounting the 44 (53%) cases of peripheral regional hospital referrals. Abnormal placentation and uterine atony constituted the majority of the indications for peripartum hysterectomy. Abnormal placentation included placenta praevia, accrete, increta and percreta. In this cohort with peripartum hysterectomy, previous caesarean section was strongly associated with abnormal placentation (P < 0.001, OR 11.4, 95% CI 3.6-35.8). No maternal mortality was recorded, although 63% of patients encountered complications. A planned peripartum hysterectomy resulted in significantly fewer red blood cell (P = 0.011) and platelet transfusions (P = 0.001). CONCLUSIONS: The incidence of peripartum hysterectomy recorded in our tertiary institution between 2000 and 2014 is 0.60 per 1000 births. Abnormal placentation is the commonest indication leading to severe postpartum haemorrhage requiring peripartum hysterectomy.

The changing birth suite experience for Australian medical students.

Competition with midwifery students, combined with increasing numbers of medical students, has led to declining standards of clinical experience in the birth suite for our senior medical students. In the Australian setting, increased and improved communication and collaboration between the two disciplines is urgently needed to rectify the situation.

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

Correlating chemical sensitivity and basal gene expression reveals mechanism of action.

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ∼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.

Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset.

UNLABELLED: Identifying genetic alterations that prime a cancer cell to respond to a particular therapeutic agent can facilitate the development of precision cancer medicines. Cancer cell-line (CCL) profiling of small-molecule sensitivity has emerged as an unbiased method to assess the relationships between genetic or cellular features of CCLs and small-molecule response. Here, we developed annotated cluster multidimensional enrichment analysis to explore the associations between groups of small molecules and groups of CCLs in a new, quantitative sensitivity dataset. This analysis reveals insights into small-molecule mechanisms of action, and genomic features that associate with CCL response to small-molecule treatment. We are able to recapitulate known relationships between FDA-approved therapies and cancer dependencies and to uncover new relationships, including for KRAS-mutant cancers and neuroblastoma. To enable the cancer community to explore these data, and to generate novel hypotheses, we created an updated version of the Cancer Therapeutic Response Portal (CTRP v2). SIGNIFICANCE: We present the largest CCL sensitivity dataset yet available, and an analysis method integrating information from multiple CCLs and multiple small molecules to identify CCL response predictors robustly. We updated the CTRP to enable the cancer research community to leverage these data and analyses.

Toward performance-diverse small-molecule libraries for cell-based phenotypic screening using multiplexed high-dimensional profiling.

High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.

Vitamin D concentrations in pregnant women with diabetes attending for antenatal care in Far North Queensland.

Serum concentrations of vitamin D were measured in 101 pregnant women with diabetes, both pre-existing and gestational, who attended for antenatal care in Cairns Base Hospital. Eighty-two (81.2%) had sufficient concentrations of vitamin D, 12 (11.9%) had levels indicating insufficiency and 7 (6.9%) were deficient. These findings contrast with those in the general population of pregnant women in the region, among whom 93.1% have been shown to have sufficient levels. The study contributes to the ongoing debate around the need for universal antenatal vitamin D screening in Australia.

An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules.

The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene ß-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.