Select Applications

Some newer applications of FAC-MS have been described, which suggest that the technology has a strong role to play in the drug discovery process for an extremely broad range of molecular interactions.

The method has been applied as a ''global kinase binding assay'', in which both ATP and substrate binding sites could be monitored in the same experiment using indicator methods described above (Fig. 6.15) [32]. A similar FAC-MS assay was developed around the anticancer target kinase, EphB2 and the data compared to an ELISA for the same protein [33]. In this work, a series of known kinase inhibitors were interrogated by both methods using the indicator approach focusing on the ATP binding site, and the correlation between FAC and ELISA data was strong.

The method has been used as an adjunct to virtual library screening, in which a set of compounds was screened against a pharmacophoric model of the ATP

Fig. 6.15 FAC-MS chromatograms of dual indicators for protein kinase Ca [32]. (a) In the chromatograms, the red lines correspond to a void marker, the blue lines correspond to the substrate-site indicator chelerythrine chloride and the magenta lines correspond to the ATP-site indicator PD153035. Arrows indicate respective shifts when screening. (b) WHI-P180, an ATP-site binder, (c) inhibitor peptide 19-36, a substrate-site binder and (d) both WHI-P180 and inhibitor peptide 1936. Adapted with permission from the American Chemical Society.

Fig. 6.15 FAC-MS chromatograms of dual indicators for protein kinase Ca [32]. (a) In the chromatograms, the red lines correspond to a void marker, the blue lines correspond to the substrate-site indicator chelerythrine chloride and the magenta lines correspond to the ATP-site indicator PD153035. Arrows indicate respective shifts when screening. (b) WHI-P180, an ATP-site binder, (c) inhibitor peptide 19-36, a substrate-site binder and (d) both WHI-P180 and inhibitor peptide 1936. Adapted with permission from the American Chemical Society.

binding site in EphB2 [34]. This serves to ''weed out'' compounds that do not conform to the model and thus would likely show a reduced probability of generating hits in a real screen. In this example, the authors reduced their library from an initial 50 542 unique compounds to 468, which were then acquired and screened via FAC-MS. These were then binned into mixtures of nine compounds each and screened using a simple indicator analysis. While this method only provides a bulk measurement of inhibition (i.e. does not discriminate between many weak binders and single stronger binders), the use of very small mixture sizes makes this approach feasible. Even so, it was shown that over half of the mixtures generated measurable indicator shifts, and that larger mixtures may have rendered this particular indicator method ineffective. Nevertheless, mixtures with low to moderate hit rates would benefit from this sort of prescreen [10]. Four mixtures generating the largest shifts were further investigated - individual components were screened using a single-point indicator experiment, and the results correlated well with ELISA data for the individual hits.

Nonprotein screening exercises have also been developed around the FAC method. We have demonstrated that affinity constructs can be formulated around immobilized 16S A-site rRNA, and used to screen aminoglycoside-binding to the A-site domain. An online experiment was performed, where it could be shown that neamine, lividomycin and paromomycin eluted in the expected order based on previous studies [35]. Interestingly, FAC columns constructed around immobilized RNA could not be regenerated after ligand binding, requiring that a new column be used for each experiment. With column volumes of 800 nl, this is not a difficulty as many columns can be prepared from a single batch of stationary phase.

FAC-MS has been used recently in antiviral development projects to discover two small molecule natural products inhibiting the entry of SARS-Coronavirus into Vero-E6 cells [26]. A range of Chinese herbs (121 different species) were extracted in 85% ethanol and screened via online FAC-MS (using an electrospray TOF instrument), through a column containing the SARS S2 protein. The authors estimate that @130 compounds with Kds under 10 mM were discovered by FAC-MS, and of these hits, two molecules (luteolin and tetra-O-galloyl-b-D-glucose) exhibited low micromolar EC50s in an infection assay using a pseudo-typed virus, correlating well with their highest binding strength in the FAC assay. These two molecules exhibit an activity far superior to glycyrrhizin, another small molecule recently reported to exhibit antiSARS-CoV activity [36].

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment