Natural product libraries to accelerate the high-throughput discovery of therapeutic leads.

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

HALO384: a halo-based potency prediction algorithm for high-throughput detection of antimicrobial agents.

A high-throughput (HT) agar-based halo assay is described, which allows for rapid screening of chemical libraries for bioactivity in microorganisms such as yeast and bacteria. A pattern recognition algorithm was developed to identify halo-like shapes in plate reader optical density (OD) measurements. The authors find that the total growth inhibition within a detected halo provides an accurate estimate of a compound's potency measured in terms of its EC(50). The new halo recognition method performs significantly better than an earlier method based on single-point OD readings. An assay based on the halo algorithm was used to screen a 21,120-member library of drug-like compounds in Saccharomyces cerevisiae, leading to the identification of novel bioactive scaffolds containing derivatives of varying potencies. The authors also show that the HT halo assay can be performed with the pathogenic bacterium Vibrio cholerae and that liquid culture EC(50) values and halo scores show a good correlation in this organism. These results suggest that the HT halo assay provides a rapid and inexpensive way to screen for bioactivity in multiple microorganisms.

Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries.

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors from the NCI repository. One is the potent but non-selective michellamine B, a natural product, anti-viral agent. The other four compounds were selective inhibitors against 12-hLO, with three being synthetic compounds and one being alpha-mangostin, a natural product, caspase-3 pathway inhibitor. In addition, a selective inhibitor was isolated from the UCSC-MEL (neodysidenin), which has a unique chemical scaffold for a hLO inhibitor. Due to the unique structure of neodysidenin, steady-state inhibition kinetics were performed and its mode of inhibition against 12-hLO was determined to be competitive (K(i)=17microM) and selective over reticulocyte 15-hLO-1 (K(i) 15-hLO-1/12-hLO>30).

Accelerating the discovery of biologically active small molecules using a high-throughput yeast halo assay.

The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.