Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors.

Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

An assessment of Motorola CodeLink microarray performance for gene expression profiling applications.

DNA microarrays enable users to obtain information on differences in transcript abundance on a massively parallel scale. Recently, however, data analyses have revealed potential pitfalls related to image acquisition, variability and misclassifications in replicate measurements, cross-hybridization and sensitivity limitations. We have generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. Together, we have used these tools to optimize performance in an expression profiling study. We demonstrate three significant advantages of the Motorola CodeLink platform: sensitivity of one copy per cell, coefficients of variation of 10% in the hybridization signals across slides and across target preparations, and specificity in distinguishing highly homologous sequences. Slides where oligonucleotide probes are spotted in 6-fold redundancy were used to demonstrate the effect of replication on data quality. Lastly, the differential expression ratios obtained with the CodeLink expression platform were validated against those obtained with quantitative reverse transcription-PCR assays for 54 genes.

Oligodeoxyribonucleotide probe accessibility on a three-dimensional DNA microarray surface and the effect of hybridization time on the accuracy of expression ratios.

BACKGROUND: DNA microarrays are now routinely used to monitor the transcript levels of thousands of genes simultaneously. However, the array fabrication method, hybridization conditions, and oligodeoxyribonucleotide probe length can impact the performance of a DNA microarray platform. RESULTS: We demonstrate solution-phase hybridization behavior of probe:target interactions by showing a strong correlation between the effect of mismatches in probes attached to a three dimensional matrix of a microarray and solution-based, thermodynamic duplex melting studies. The effects of mismatches in the probes attached to the microarray also demonstrate that most, if not all, of the oligodeoxyribonucleotide is available for hybridization. Kinetic parameters were also investigated. As anticipated, hybridization signals increased in a transcript concentration-dependent manner, and mismatch specificity increased with hybridization time. Unexpectedly, hybridization time increased the accuracy of fold changes by relieving the compression observed in expression ratios, and this effect may be more dramatic for larger fold changes. CONCLUSIONS: Taken together, these studies demonstrate that a three-dimensional surface may enable use of shorter oligodeoxyribonucleotide probes and that hybridization time may be critical in improving the accuracy of microarray data.