Cytochrome P450 2C epoxygenases mediate photochemical stress-induced death of photoreceptors.

Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.

Novel N-benzoyl-2-hydroxybenzamide disrupts unique parasite secretory pathway.

Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3ß, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.

Clinical validation of the Tempus xO assay.

We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification.

Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.