Design and synthesis of novel N-sulfonyl-2-indole carboxamides as potent PPAR-gamma binding agents with potential application to the treatment of osteoporosis.

The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-gamma) are reported. Chemical optimization of the series led to the identification of 4q (IC(50)=50 nM) as a potent binding agent of PPAR-gamma. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.

MALDI-TOF MS as a label-free approach to rapid inhibitor screening.

Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by time-consuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC(50) curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were <7% RSD-a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development.

Comparison of high throughput screening technologies for luminescence cell-based reporter screens.

As higher density formats become more and more common in HTS labs, the expectations for maintaining faster, lower cost screens puts great pressure on traditional 96-well screens. In some cases higher density formats are not compatible with the assay. This seems especially true in cell-based assays. In our case, the nature of the cells' response forced us to remain in 96-well plates. In this paper, we describe the development of a luminescence reporter assay and its performance in two detection modes, flash and glow. The advantages in cost and throughput for each technique are explored, along with automation considerations. An additional new technology, the use of pins for low-volume transfers, is also briefly described because of its dramatic effect on our screen's throughput. However, it will be more thoroughly presented in a future publication. Comparing the technologies available for HTS aids in designing automated systems that meet the unique needs of each assay.