Automated multiple ligand screening by frontal affinity chromatography-mass spectrometry (FAC-MS).

High-throughput screening (HTS) efforts to discover "hits" typically rely on the large-scale parallel screening of individual compounds with attempts to screen mixtures of compounds typically and, unfortunately, giving rise to false positives and false negatives due to the nature of the HTS readout (% inhibition/activation above a defined threshold) that makes deconvolution virtually intractable. Bioaffinity screening methods have emerged as an alternative or orthogonal method to classic HTS. One of these methods, frontal affinity chromatography coupled to mass spectrometry detection (FAC-MS), although still a relatively new technique, is turning out to be a viable screening tool. However, to push FAC-MS more to the forefront as a moderate primary HTS system (or a secondary screening assay), automation needs to be addressed. An automated FAC-MS system is described using 2 columns containing immobilized hERbeta, whereby while 1 column is being regenerated, the other is being used. The authors are extrapolating that in a continuous 24-h operation, the number of ligands screened could potentially approach 10,000. In addition, preliminary structure-activity relationship binding information (typically not seen in early primary HTS) can be obtained by observing the rank order of the library members in the various mixtures.

Frontal affinity chromatography with MS detection (FAC-MS) in drug discovery.

The emergence of a relatively new technique resulting from a combination of frontal affinity chromatography coupled with MS detection (FAC-MS) has extended the capabilities of MS in drug discovery and development. Its application in a broad range of biological systems, together with its label-free operation, relatively high throughput, ability to rank ligands and determine Kd, makes FAC-MS a universal tool enabling convenient and efficient screening in the identification of new potential drug leads. Here we will highlight FAC-MS screening studies and discuss where it can be applied in evaluating multiple protein-binding sites, protein-protein interactions and inactive proteins, and also in determining selectivity.

Frontal affinity chromatography with MS detection of EphB2 tyrosine kinase receptor. 2. Identification of small-molecule inhibitors via coupling with virtual screening.

We have integrated two complementary methods, high-throughput virtual screening with a "high-content" wet screening technique based on frontal affinity chromatography with mass spectrometry detection (FAC-MS), for identification of hits against the erythropoietin-producing hepatocellular B2 (EphB2) receptor tyrosine kinase domain. Both an EphB2-directed virtual screen combining docking and scoring and a kinase-directed pharmacophore search strategy were used to identify a compound set enriched in bioactive compounds against EphB2. The coupling of virtual screening methodologies with FAC-MS is a unique hybrid approach that can be used to increase the efficacy of both hit discovery and optimization efforts in drug discovery and has successfully identified hits, in particular 19a (36% shift, IC(50) = 5.2 microM, K(d) = 3.3 microM), as inhibitors for EphB2, a potential cancer target.

In vitro evaluation of the anti-estrogenic activity of hydroxyl substituted diphenylnaphthyl alkene ligands for the estrogen receptor.

There is still a need for additional scaffolds to further explore tissue selectivity and improving efficacy of selective estrogen receptor modulators (SERMs). A series of hydroxyl substituted diphenylnaphthyl alkene ligands for the two estrogen receptors are described that arose from an initial de novo designed diphenylnaphthyl propylene ligand 1. All compounds gave K(i)s under 10 nM when assayed in the presence of ERalpha. Generally these compounds had very high affinity for both ER isotypes. Moving the hydroxyl group on naphthalene from the 6- to the 5-position of the alpha-naphthalene attached compounds (6b and 6e vs 6c and 6f) had little affect on ER binding nor did altering the position of the naphthalene attachment (alpha or beta) to the alkene moiety. In transfection assays none of the compounds displayed agonistic activity in the absence of E(2). In MCF-7 proliferation assays 6a-d, 6f and 12a-c successfully abrogated E(2) stimulation and resulted in greater than 50% inhibition at 1 microM, a level of efficacy similar to that obtained when the cells were treated with raloxifene. Our results show that this new class of SERMs are good candidates for further study as therapeutic agents for the treatment of breast cancer and osteoporosis.

Global kinase screening. Applications of frontal affinity chromatography coupled to mass spectrometry in drug discovery.

Utilizing frontal affinity chromatography with mass spectrometry detection (FAC-MS), we have identified novel applications in the discovery of small-molecule hits to protein targets that are difficult if not impossible to accomplish using traditional assays. We demonstrate for the first time an ability to distinguish between competitive ligands for the ATP and substrate sites of protein kinase C independently in the same experiment and show that ATP competitive ligands using a functionally inactive receptor tyrosine kinase can be identified. This ability of FAC-MS to simultaneously monitor binding at the ATP and substrate binding sites, as well as measure ligand binding to both active and inactive kinases, suggests that FAC-MS can be used as a "global kinase binding assay".

Frontal affinity chromatography with MS detection of EphB2 tyrosine kinase receptor. 1. Comparison with conventional ELISA.

FAC-MS offers a convenient method for measuring the relative binding strengths of ligands in a mixture and enables a rapid ranking and identification of ligands in the mixture as potential hits against immobilized targets. Using immobilized EphB2 receptor tyrosine kinase as the target and known kinase inhibitors, the results of FAC-MS screening (% shift) have been shown to correlate with the binding constant, K(d), and with IC(50) results from the more traditional ELISA assay. Therefore, since FAC-MS can accommodate a wide variety of target proteins, its applications could play a broad role in drug discovery not only at the hit discovery stage but also during the subsequent more rigorous screening at the hit-to-lead and lead optimization stages.

Synthesis and evaluation of a novel nonsteroidal-specific endothelial cell proliferation inhibitor.

The identification of agents with specific antiproliferative or cytostatic activity against endothelial cells has significant value for the treatment of pathologies associated with angiogenesis, including solid tumors. Here, we describe a novel substituted dibenzo[b,d]pyran-6-one scaffold, exemplified by structures 9a and 10, and report preliminary in vitro activity data indicating that this scaffold is a promising lead for the development of specific inhibitors of endothelial cell proliferation.

De novo design, synthesis, and evaluation of novel nonsteroidal phenanthrene ligands for the estrogen receptor.

Although there are many estrogen receptor antagonists with improved tissue selectivity profiles compared with tamoxifen, optimal tissue selectivity has not yet been demonstrated. As such there is still a need for additional diversity and new chemical scaffolds to allow for exploration of improved tissue selectivity. Here, we describe the discovery of a novel phenanthrene scaffold for estrogen receptor ligands utilizing a ligand based de novo design approach. The nanomolar binding of phenanthrenes, 12b,c, 14b,c, and 15 against human recombinant ER(alpha) indicates that our ligand based de novo design approach was successful. From a gene transfection assay, 12b,c, 14b,c, and 15 displayed only antagonistic activity with no observable agonistic activity. The alkyl 9,10-dihydrophenanthrene 16 (presumably a racemic mixture) was a substantially more potent ER binder than the phenanthrenes. It also displayed only antagonistic activity and was effective at inhibiting estradiol stimulated MCF-7 cell proliferation. These results demonstrate that this phenanthrene (and 9,10-dihydrophenanthrene) scaffold warrants further study as potential selective estrogen receptor modulators and/or pure antiestrogens.

De novo design, synthesis and evaluation of a non-steroidal diphenylnaphthyl propylene ligand for the estrogen receptor.

There is still a strong need for additional diversity and new chemical scaffolds to allow for the exploration of improved tissue selectivity and finding better selective estrogen receptor modulators (SERMs). Using a de novo design technology a diphenylnaphthyl propylene scaffold, exemplified by (E)-9b, with ER antagonist activity has been generated. It was prepared by alkylating 1-[4-methoxyphenyl)-2-(4-(2-chloroethoxy)phenyl]-1-propanone under metal halogen exchange conditions with 1-iodo-6-methoxy-naphthalene. Following dehydration and cleavage of the methoxy groups, (E)-9b was formed by displacement of the chloro group with pyrrolidine. (E)-9b binding to ER generated calculated K(i) values of 3.7 nM for hER(alpha) and 72 nM for hER(beta). The antagonism of (E)-9b was demonstrated in cell transfection assays using the ERE from the vitA2 promotor and the natural ER-responsive pS2 promotor. With increasing concentrations of (E)-9b, the E(2)-dependent response was efficiently inhibited demonstrating that (E)-9b could function as an anti-estrogen in these assays. Interestingly, ER(alpha) activity was inhibited even below basal levels suggesting that ligand-independent activity of ER(alpha) was also inhibited. Computational docking studies suggest that the placement of the hydroxyl group on the naphthalene group may not be optimal and we are currently exploring additional analogues.