Discovery of a ZIP7 inhibitor from a Notch pathway screen.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.

Bile acid analogues are activators of pyrin inflammasome.

Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.

Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

Discovery and Structure-Based Design of Inhibitors of the WD Repeat-Containing Protein 5 (WDR5)-MYC Interaction.

WDR5 is a critical chromatin cofactor of MYC. WDR5 interacts with MYC through the WBM pocket and is hypothesized to anchor MYC to chromatin through its WIN site. Blocking the interaction of WDR5 and MYC impairs the recruitment of MYC to its target genes and disrupts the oncogenic function of MYC in cancer development, thus providing a promising strategy for the treatment of MYC-dysregulated cancers. Here, we describe the discovery of novel WDR5 WBM pocket antagonists containing a 1-phenyl dihydropyridazinone 3-carboxamide core that was identified from high-throughput screening and subsequent structure-based design. The leading compounds showed sub-micromolar inhibition in the biochemical assay. Among them, compound 12 can disrupt WDR5-MYC interaction in cells and reduce MYC target gene expression. Our work provides useful probes to study WDR5-MYC interaction and its function in cancers, which can also be used as the starting point for further optimization toward drug-like small molecules.

Jet Ventilation in the Pregnant Patient with Airway Stenosis: Surgical Safety and Outcomes.

OBJECTIVE: To review pregnancy outcomes and the safety of jet ventilation use in the gravid patient undergoing surgical airway intervention. METHODS: A multi-institutional retrospective review of medical records was performed to identify women who underwent low-frequency jet ventilation during pregnancy for surgical treatment of airway stenosis. Postoperative complications were noted, and patients were interviewed regarding pregnancy outcomes. RESULTS: Six women were included in this series. No immediate complications relating to anesthesia or surgical intervention were noted in five of the six women. One patient with a well-known history of uncontrolled seizures experienced seizure activity postoperatively. One patient returned to the operating room at a later date for debridement of tracheal crusts. Five mothers delivered via cesarean section and one via spontaneous vaginal delivery. The mean gestation age was 37.3 weeks. One of the six infants delivered prematurely and three were delivered at low birth weight. Three of the six infants required elevated care immediately post-delivery but, at present, all are in good health. CONCLUSION: Low-frequency jet ventilation and surgical management of airway stenosis should be recognized as a safe treatment option in the gravid patient. Surgical intervention should not be delayed in patients with severe symptoms, particularly given the potential risk associated with prolonged corticosteroid use. LEVEL OF EVIDENCE: 4.

Systematic Chemogenetic Library Assembly.

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners.

Neutrophil/lymphocyte ratio is associated with survival in synovial sarcoma.

BACKGROUND: Synovial sarcoma is a rare malignant soft tissue tumor, more common in adolescents and young adults and entails a poor prognosis. Several good prognostic factors have been well established such as age less than 25, size less than 5 cm and absence of a poorly differentiated component. Inflammation has a well-established role in tumor proliferation and survival. The aim of this study was to investigate the prognostic significance of the neutrophil/lymphocyte ratio (NLR) in a large cohort of synovial sarcoma patients. METHODS: Retrospective study of 169 consecutive patients. We analyzed the relation of preoperative NLR on disease-free survival (DFS) and overall survival (OS) using Kaplan-Meier curves and Cox proportional models. RESULTS: Of the 169 patients included, there were 90(53.3%) females and 79(46.7%) males. Median age was 32yo (11-73). Median survival was 34.1 and mean disease-free survival was 21.4 months. Mean tumor size was 12.5 cm (1.2-77 cm). Applying receiver operating curve analysis, we determined a cut-off value of 3.5. In univariate and multivariate analysis, increased NLR was significantly associated with poor OS. A <3.5 NLR was an independent prognostic factor in all stages (p = 0.002). CONCLUSIONS: NLR >3.5 was found to be a reliable prognostic factor in this cohort. Given its widespread availability, we believe it's use in clinical practice and further clinical trials should be considered.

A Photoaffinity Labeling-Based Chemoproteomics Strategy for Unbiased Target Deconvolution of Small Molecule Drug Candidates.

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.

Discovery of 2-Pyridylpyrimidines as the First Orally Bioavailable GPR39 Agonists.

The identification of highly potent and orally bioavailable GPR39 agonists is reported. Compound 1, found in a phenotypic screening campaign, was transformed into compound 2 with good activity on both the rat and human GPR39 receptor. This compound was further optimized to improve ligand efficiency and pharmacokinetic properties to yield GPR39 agonists for the potential oral treatment of type 2 diabetes. Thus, compound 3 is the first potent GPR39 agonist (EC50s ≤ 1 nM for human and rat receptor) that is orally bioavailable in mice and robustly induced acute GLP-1 levels.

1-amino-4-benzylphthalazines as orally bioavailable smoothened antagonists with antitumor activity.

Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.

The identification of pyrimidine-diazabicyclo[3.3.0]octane derivatives as 5-HT2C receptor agonists.

The 5-HT2C receptor has been implicated in the regulation of appetite. As such, small molecule agonists to this receptor may serve as novel therapies to combat obesity. We describe here the identification, synthesis, and SAR of a 5-HT2C agonist from a unique pyrimidine-diazabicyclo[3.3.0]octane series. This compound displayed good potency at the 5-HT2C receptor, modest selectivity relative to other 5-HT2 receptors, and was efficacious in an acute feeding study in rats.

The design and synthesis of a tricyclic single-nitrogen scaffold that serves as a 5-HT2C receptor agonist.

5-HT2C agonists have shown efficacy in limiting food consumption and thus may serve as an important drug class in combating obesity. We describe the design and synthesis of a novel tricyclic single-nitrogen scaffold that was used to produce 5-HT2C agonists. SAR was developed around this chemotype and compounds were identified that were potent (Ki<15 nM) and selective relative to other 5-HT2 receptors. The most promising compound displayed a good pharmacokinetic profile in multiple animal species, and was efficacious in an acute feeding study in rats.