Dependence on the Pyrimidine Biosynthetic Enzyme DHODH Is a Synthetic Lethal Vulnerability in Mutant KRAS-Driven Cancers.

Activating KRAS mutations are major oncogenic drivers in multiple tumor types. Synthetic lethal screens have previously been used to identify targets critical for the survival of KRAS mutant cells, but their application to drug discovery has proven challenging, possibly due in part to a failure of monolayer cultures to model tumor biology. Here, we report the results of a high-throughput synthetic lethal screen for small molecules that selectively inhibit the growth of KRAS mutant cell lines in soft agar. Chemoproteomic profiling identifies the target of the most KRAS-selective chemical series as dihydroorotate dehydrogenase (DHODH). DHODH inhibition is shown to perturb multiple metabolic pathways. In vivo preclinical studies demonstrate strong antitumor activity upon DHODH inhibition in a pancreatic tumor xenograft model.

Preparation and optimization of pyrazolo[1,5-a]pyrimidines as new potent PDE4 inhibitors.

A new series of pyrazolo[1,5-a]pyrimidines exemplified by compound 1, has been identified with moderate activity (IC50=165nM), following GSK256066 rescaffolding. Compound 1 optimization at positions 2, 3, 6 and 7 gave compound 10 with high in vitro activity (IC50=0.7nM). Modeling studies based on the PDB structure 3GWT with compound 5 showed the expected overlay with the carboxamide, the aryl moiety and the sulfone. Cyclisation of the primary amide to the 5 position of the pyrazolo[1,5-a]pyrimidines scaffold afforded compounds 15 and 16 with 200-fold enhancement in activity and cellular potency.

Dystrophin Dp71 gene deletion induces retinal vascular inflammation and capillary degeneration.

We have previously shown that the deletion of the dystrophin Dp71 gene induces a highly permeable blood-retinal barrier (BRB). Given that BRB breakdown is involved in retinal inflammation and the pathophysiology of many blinding eye diseases, here we investigated whether the absence of Dp71 brings out retinal vascular inflammation and vessel loss by using specific Dp71-null mice. The expression of vascular endothelial growth factor (VEGF), quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay methods, was higher in the retina of Dp71-null mice than in wild-type mice. In contrast, no differences were observed in VEGFR-2 and tumor necrosis factor-α expression. Moreover, mRNA expression of water channel, aquaporin 4 (AQP4) was increased after Dp71 deletion. The Dp71 deletion was also associated with the overexpression of intercellular adhesion molecule 1, which is expressed on endothelial cells surface to recruit leukocytes. Consistent with these findings, the total number of adherent leukocytes per retina, assessed after perfusion with fluorescein isothiocyanate-conjugated concanavalin A, was increased in the absence of Dp71. Finally, a significant increase in capillary degeneration quantified after retinal trypsin digestion was observed in mice lacking Dp71. These data illustrate for the first time that the deletion of Dp71 was associated with retinal vascular inflammation, vascular lesions with increased leukocyte adhesion and capillary degeneration. Thus, dystrophin Dp71 could play a critical role in retinal vascular inflammation disease, and therefore represent a potential therapeutic target.

Tricyclic series of heat shock protein 90 (Hsp90) inhibitors part I: discovery of tricyclic imidazo[4,5-c]pyridines as potent inhibitors of the Hsp90 molecular chaperone.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed after a low throughput screen (LTS) of a focused library containing approximately 21K compounds selected by virtual screening. The initial [1-{3-H-imidazo[4-5-c]pyridin-2-yl}-3,4-dihydro-2H-pyrido[2,1-a]isoindole-6-one] (1) compound showed moderate activity (IC(50) = 7.6 µM on Hsp82, the yeast homologue of Hsp90). A high-resolution X-ray structure shows that compound 1 binds into an "induced" hydrophobic pocket, 10-15 Å away from the ATP/resorcinol binding site. Iterative cycles of structure-based drug design (SBDD) and chemical synthesis led to the design and preparation of analogues with improved affinity. These optimized molecules make productive interactions within the ATP binding site as reported by other Hsp90 inhibitors. This resulted in compound 8, which is a highly potent inhibitor in biochemical and cellular assays (K(d) = 0.35 nM on Hsp90; IC(50) = 30 nM on SKBr3 mammary carcinoma cells) and in an in vivo leukemia model.

Direct measurement of multiple mRNAs in nerve growth factor-induced PC12 cells using electrophoretic tags to monitor biomarkers of neuronal differentiation in 96-well format.

Pheochromocytoma-12 (PC12) cells recapitulate the program of neuronal differentiation by developing neurites after about 12 days of nerve growth factor (NGF) treatment. This model can be used to evaluate the neuroprotective/neurotrophic effect of compounds. Specific mRNAs such as cfos and c-jun are early biomarkers of the irreversible commitment into the differentiation program as they appear after only 30-40 min of NGF treatment. Monitoring the level of these mRNAs instead of the neurite outgrowth dramatically reduces the time needed to identify the drug potential of compounds. The electrophoretic tags, or eTag reporters (ACLARA Biosciences, Inc., Mountain View, CA), are a new class of fluorescent reporters that have unique migration properties in capillary electrophoresis, which allows for their separation and identification. (The eTag Multiplex Invader Assay and products incorporate Invader technology and Cleavase enzyme licensed for use from Third Wave Technologies, Inc. [Madison, WI] for multiplexed gene expression applications.) Each eTag molecule used begins as a phosphoramidite that is incorporated into a specific oligonucleotide using standard oligonucleotide synthesis procedures. A set of distinct probes labeled with different eTag molecules can then be mixed together to simultaneously quantify the levels of different mRNAs from the same sample. When compared to existing methods for measuring multiplexed gene expression from the same sample, the eTag assay allows a direct quantification of the mRNA from cells without any extraction/purification and still provides multiplexing capability, high sensitivity, miniaturization, and reproducibility compatible with medium-throughput screening methods. The eTag technology was used to simultaneously measure the level of expression of four mRNAs-c-fos, c-jun, c-myc, and gapdh-in NGF-treated PC12 cells in a standard 96-well format. The experimental data shown here demonstrate the use of eTag technology as a new screening tool, which uniquely combines robustness, sensitivity, multiplexing capability, and direct measurement of mRNA without any sample preparation steps, such as RNA extraction/purification or a reverse transcription step.