Screening Ligandreceptor Binding In The Detection Of Novel Natural Products

Technologies that focus on the direct interaction of molecules such as natural products with molecular targets can serve to reduce the cycle time inherent in bioassay-directed fractionation and identification. The use of affinity-based

FIGURE 6.4 Schematic representation of the high-throughput methods used by Eldridge et al. (Reprinted with permission from Anal. Chem (see ref 66))

techniques coupled with mass-spectral detection for screening is an area of active research and has been the subject of recent review [68]. One such technique is the "high resolution screening" (HRS) MS-based technique described by Schobel et al. [69]. In this example, plant extracts were initially screened in an estrogen receptor-binding fluorescence-enhancement assay that used the fluorescent ligand coumesterol. Extracts that contained ligands which bind to the estrogen receptor were detected by a reduction in coumesterol fluorescence. Binding activity was then confirmed with competitive fluorescence polarization, functional activity, and transcriptional activation assays. Natural product extracts that were confirmed to be active in all these assays were next processed in the HRS MS mode. After injection and chromatographic separation of the extract, the coumesterol + estrogen receptor solution was infused into the effluent stream of the LC. A decrease in fluorescence from the coumes-terol/estrogen receptor was used to indicate activity. Active peaks were directed to the mass spectrometer where MS and MS/MS spectra were acquired in data-dependent scan mode. The LC retention time, molecular weight, and MS/MS spectra were then used to perform database searches to elucidate the structures of the bioactive compounds. In this fashion, molecular weight information on bioactive compounds as well as MS/MS spectra to support structure elucidation was obtained in a single analysis. Limitations of this technique include various matrix effects (i.e., fluorescence quenching, absorbance, light scattering, nonspecific binding between matrix compounds and target), and nontrivial statistical analysis. Nonetheless, reduced cycle time was demonstrated through this on-line ligand-receptor binding MS-based approach.

Another direct method for the detection of active natural products has centered on the binding of ligands to ribonucleic acid (RNA) targets [70]. One such technique is referred to as multitarget affinity/specificity screening (MASS). This approach is based on the identification of library members bound to a macromolecular target with the direct measurement of the accurate mass of the target-ligand complex. The technique uses the resolving power and mass accuracy of FTMS, allowing the simultaneous screening of multiple targets against multiple ligands. High-resolution accurate mass scans of RNA targets are first acquired without the addition of a screening mixture (extract). The extract is then added, and the mass spectrum of the mixture is evaluated for mass shifts that indicate binding of the ligand to a target. The number of targets screened is limited by the complexity of the target spectrum, but can utilize as many as 10 targets simultaneously, or fewer targets with increasingly complex ligand mixtures. Interestingly, the screening mixture concentration can be modified to select for weak (^M) to strong (nM) binders. Not surprisingly, with this technique data interpretation is complex and presents a significant challenge.

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