urge working with expensive assay formats or coy targets.such the drug disc ss during the last ecade. Natural pure compounds,fraction and extract ne screening fommat The Discovery Process of Bioactive Natural Products cations In rece ch of s ple appr eening crude ore-pu could not match with thesh HPLC-DAD.MS and -NMR h opened entire rt tars here te rovide minute prod ture to pure t.Fuund 16 on-line wit urate IC shed using HPLC-CD indispe recent mass is ning importan Recent devel NM ngletons, fac that puts them cal stab mak synthetic librarie crystall sub-mg ing increasingly the to the comp which While analysis,purification and str cture eluci commo ispla nins,for e Tan tight com with n ugh over the last decade,tracking bioactivity in complex ma ult in most able tha Detergent-like compounds havea tn methods to The a nclud from which are common in nts has stre tomve nd such a and ss req ubstantia m and ous prore r2111t raphic sep meta chelat steps of preparative chroma akdown product While this proc lure has led t a e to interfere the readout in assays based on light be g slow and stly the se n HTS and a slow labour-intensiv The decon of b vity e of the dereplication known or vise stin ib of com nds an other ab onation 12.1 nd the of me drug discovery limi ting the d for faster and m Pure cup es oughpu onment.Pr bably th greatest cha enge titute the hug ctural divers nd in extr iology to corre clate che are likely found in Var on the othe nd lea erable incr of the for t is of macrom ber of samples to be an isst teractions, an he 900 Current Organic Chemistry, 2006, Vol. 10, No. 8 Potterat and Hamburger recent decline of interest observed in the pharmaceutical industry is, in part, due to the accelerated transformation of the drug discovery process during the last decade. Natural products have been facing major obstacles to fit into this new drug research environment. The advent of combinatorial chemistry and high throughput screening [10] in the 1990s enabled the testing of hundreds of thousands samples within a few weeks. This paradigm shift had major implications for natural product research. The classical and historically successful approach of screening crude or pre-purified extracts followed by several iterative steps of activity guided fractionation could not match with the short target cycle times in HTS, where testing capacities are only provided for a limited time window. A further complication is that natural product hits must go from mixture to pure compounds with enough time left for hit-to-lead assessment. Full structural information and accurate IC50 data are indispensable to compete with synthetic compounds in the lead selection phase. Hit clustering is gaining importance for establishing structure-activity relationship early in the lead selection process. Natural product hits are often observed as singletons, a fact that puts them at a disadvantage in comparison to compound families typically encountered in synthetic libraries. A further issue with extract screening is the comparatively high number of false positives which are due to the common presence of compounds which display unspecific activities or interfere with the assay format. Tannins, for example, form tight complexes with metal ions and with a wide array of proteins and polysaccharides. This leads to false-positive result in most assays involving a purified protein. Detergent-like compounds have a tendency to disrupt membranes and produce misleading results in cell￾based assays. Examples include widely occuring plant metabolites such as saponins, and fatty acids and panosialins, which are common in streptomycetes. Compounds such as polyenes and polyethers often display general cytotoxicity and may produce false results in cell-based assays. Strong metal chelators are susceptible to react with assay components, e.g. when nickel beads are used as linkers. UV quenchers such as the chlorophyll breakdown product phaeophorbide A, and autofluorescent compounds are prone to interfere with the readout in assays based on light measurement [11]. Confronted to unrealistic hit rates in HTS and a slow and labour-intensive deconvolution process, many pharmaceutical companies have switched from extract screening to prefractionated extracts or pure compounds libraries [12]. Large collections of compounds and semi￾purified fractions have been generated using parallel fractionation and purification technology [12-15]. While these methods have the undeniable advantage of considerably reducing or even eliminating the time￾consuming follow-up process, they also have some intrinsic drawbacks. Pure compound libraries will never be a full substitute for the huge structural diversity found in extracts. Trace components which are, in principle, as promising as major constituents, are likely not found in such collections. The splitting of an extract into a large number of fractions, on the other hand, leads to a considerable increase of the number of samples to be screened. This can be an issue when working with expensive assay formats or costly targets, such as recombinant proteins. A well-balanced combination of pure compounds, fractions and extracts, and a differential use thereof depending on the target and the screening format, appears to be the most promising approach. The Discovery Process of Bioactive Natural Products In recent years, natural product research has become a technology-driven process. The impact of HPLC-coupled spectroscopy has been tremendous. The concerted use of HPLC-DAD, -MS and -NMR has opened entirely new possibilities for the characterization of secondary metabolites in biological extracts. These techniques provide a wealth of structural information on-line with minute amounts of sample [16, 17]. Even absolute configuration of a molecule can be established using HPLC-CD [18] or HPLC-NMR after Mosher’s ester derivatization [17]. With the more recent emergence of mass spectrometry-controlled preparative HPLC [19], compound purification has also become straightforward, provided the compounds exhibit sufficient chemical stability. Recent developments in NMR probe technology and higher magnetic fields [20], and miniaturization in X-ray crystallography make structural elucidation with sub-mg amounts becoming increasingly routine. While analysis, purification and structure elucidation of natural products have experienced a technological breakthrough over the last decade, tracking bioactivity in complex matrices remains a highly challenging task. Extracts are complex mixtures. There is a continuing need for faster and more reliable methods to identify compounds that interact with therapeutic targets, with minimal interference from the multitude of chemicals present in the matrix. The classical process leading from a bioactive extract to a pharmacologically active pure constituents has always been a long and tedious process requiring substantial material amount and financial resources [21]. It consists of several consecutive steps of preparative chromatographic separation, whereby each fraction has to be submitted to suitable bioassays to track the activity ultimately to a defined pure compound. While this procedure has led to the successful isolation of many bioactive molecules, its weaknesses cannot be overlooked. Besides being slow and costly, the separation performance is poor, at least in the initial fractionation steps which are typically by open column chromatography. The loss of bioactivity in the course of the purification process is not uncommon, and there is little means for early dereplication of known or otherwise uninteresting compounds. The approach described above obviously no more matches the timelines and the workflow of modern drug discovery. There is a compelling need for faster and more effective strategies, susceptible to be implemented in a high throughput environment. Probably the greatest challenge in this context is the judicious interfacing of chemistry and biology to correlate chemical analysis with biochemical data. The development of highly sensitive and miniaturized assays provides the technological basis for that purpose. Various innovative methodologies for the analysis of macromolecule￾ligand interactions, and the on-line integration of