A multiparametric calcium signal screening platform using iPSC-derived cortical neural spheroids.

Discovery of therapeutics for neurological diseases is hampered by the lack of predictive in vitro and in vivo models. Traditionally, in vitro assays rely on engineered cell lines grown two-dimensionally (2D) outside a physiological tissue context, which makes them very amenable for large scale drug screening but reduces their relevance to in vivo neurophysiology. In recent years, three-dimensional (3D) neural cell culture models derived from human induced pluripotent stem cells (iPSCs) have been developed as an in vitro assay platform to investigate brain development, neurological diseases, and for drug screening.

Screening for modulators of neural network activity in 3D human iPSC-derived cortical spheroids.

Human induced Pluripotent Stem Cells (iPSCs) are a powerful tool to dissect the biology of complex human cell types such as those of the central nervous system (CNS). However, robust, high-throughput platforms for reliably measuring activity in human iPSC-derived neuronal cultures are lacking. Here, we assessed 3D cultures of cortical neurons and astrocytes displaying spontaneous, rhythmic, and highly synchronized neural activity that can be visualized as calcium oscillations on standard high-throughput fluorescent readers as a platform for CNS-based discovery efforts.

Functional and Mechanistic Neurotoxicity Profiling Using Human iPSC-Derived Neural 3D Cultures.

Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity.

Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.

Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC).

Chemogenetic evaluation of the mitotic kinesin CENP-E reveals a critical role in triple-negative breast cancer.

Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel.

Potent agonists of the Hedgehog signaling pathway.

A family of biaryl substituted 1,4-diaminocyclohexanamides of 3-chlorobenzothiophene-2-carboxylic acid is reported as picomolar modulators of Hedgehog protein function. SAR for the 1,4-diaminocyclohexane group is shown to be exquisitely sensitive to substitution on the 4-amino group, and SAR for the 3-chlorobenzothiophene group is highly specific. Preliminary SAR studies of the biaryl substituent led to a picomolar compound with in vivo activity.

Potent inhibitors of the hedgehog signaling pathway.

A small family of phenyl quinazolinone ureas is reported as potent modulators of Hedgehog protein function. Preliminary SAR studies of the urea substituent led to a nanomolar Hedgehog antagonist.

IFNalpha induces Fas expression and apoptosis in hedgehog pathway activated BCC cells through inhibiting Ras-Erk signaling.

Basal cell carcinoma (BCC), the most common form of human cancer, is understood to be associated with activation of the sonic hedgehog pathway, through loss-of-function mutations of tumor suppressor PTCH1 or gain-of-function mutations of smoothened. Interferon (IFN)-based therapy is quite effective in BCC treatment, but the molecular basis is not well understood. Here we report a novel mechanism by which IFNalpha mediates apoptosis in BCCs.

Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions.

The link between basal cell carcinoma (BCC) and aberrant activation of the Hedgehog (Hh) signaling pathway has been well established in humans and in mouse models. Here we report the development of assays, including two novel in vitro BCC models, which allowed us to screen for Hh inhibitors and test their validity as potential treatments for BCC. We identified a novel small molecule Hh inhibitor (CUR61414) that can block elevated Hh signaling activity resulting from oncogenic mutations in Patched-1.

Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists.

Background: The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy.

Sonic hedgehog is a potent inducer of rat oligodendrocyte development from cortical precursors in vitro.

Sonic Hedgehog (Shh) induces oligodendrocyte development in the ventral neural tube and telencephalon but its role in oligodendrocyte generation in dorsal telencephalon is debated. Transcripts for Shh and its receptor complex were detected in subventricular zone and neocortex from E17 to birth. As Shh is not yet expressed in E15 neocortex, we grew E15 cortical precursors (CP) into neurospheres in the presence of recombinant Octyl-Shh (O-Shh).