Wy824 Mw 442 011

Key: Relative Mass Spec Response Nm-iiuiltnl Afoss S/m- Ri'spim.w

Fig. 2.17 Relative non-covalent binding affinities of a variety of drug candidates to RGS4 and G alpha proteins based on the relative S/N ratios in the GPC spin column/ESI-MS assays to that of @250 pg of the respective compound (normal font) and after normalizing all the values (underlined italics font).

were found to bind to G alpha and three compounds were found to bind to RGS4 but not to G alpha. The later three compounds have the required binding properties of desirable drug candidates for inhibiting RGS4 (note that compound WY824 bound to both G alpha and RGS4 proteins).

Fig. 2.16 GPC spin column binding assay of RGS4 and G alpha proteins with WY817 (MW 450 Da). Positive ion ESI mass spectra for compound WY817, a weak binder to RGS4 protein and non-binder to G alpha protein. A miniature P6 GPC spin column was used. (A) ESI-MS response for @250 pg of reference compound WY817 (no GPC spin column used), (B) ESI-MS response for GPC spin column (P6 gel, 1 cm long, 100 |iL volume) eluate when @100 mg of WY817 were passed

through the GPC spin column. Only chemical noise is observed. (C) ESI-MS response from GPC spin column eluate when @100 mg of WY817 were incubated with 125 |iM RGS4 protein in 25 |L (70 M WY817/1 M RGS4 protein). A moderate signal is observed. (D) ESI-MS response from GPC spin column eluate when @100 mg of WY817 were incubated with 37 |iM G alpha protein in 25 mL (240 M WY817/1 M G alpha protein). No signal is observed, only chemical noise.

2.3.2.2 Amgen Primary Screens

Primary screens were performed in duplicate on mixtures of 80 compounds (1 mM per compound, 5 mM protein), where samples in each mixture were orthogonally pooled so that no two compounds that are in one well are also together in another well. Primary hits were achieved when the same compound in two wells were observed. The GPC spin-column eluates were partially resolved using a reversed-phase C18 HPLC column (Waters Xterra 2.1 x 20.0 mm) with a @1-min ballistic gradient and total cycle time of 2 min. The HPLC eluates were analyzed with a Micromass eight-way MUX interfaced to an LCT Tof ESI-MS system. Achieved throughput was @ 100 000 compounds per day.

2.3.2.3 Novartis Primary Screens

Typical Novartis screening campaigns utilized GPC spin columns constructed from 96-well plates where 400 compounds per well are assayed, utilizing 25 mL of 10 mM protein and 7 mM of each compound, all in 2% DMSO. Primary hits are detected using microbore HPLC ESI-MS with a gradient run of 10 min with tandem column switching and ion trap MS detection. Figure 2.18 is an example of a primary screen model assay for a mixture of 400 compounds with PKA protein spiked with staurosporine ([M+H]1+: m/z 467) and olomoucine ([M+H]1+: m/z 299), strong binders to the PKA protein. The automatically acquired ESI

Fig. 2.18 Raw data from a model GPC spin column/microbore HPLC ESI-MS primary screen of 400 compounds with PKA protein spiked with both staurosporine and olomoucine, known ligands of PKA. (Left) TIC, UV trace at 214 nm, and corresponding mass chromatograms for olomoucine and staurosporine. (Right) ESI mass spectra for olomoucine and staurosporine obtained from the peaks of the mass chromatograms identifying both ligands. Reprinted from reference [16] with permission from Elsevier Science.

Fig. 2.18 Raw data from a model GPC spin column/microbore HPLC ESI-MS primary screen of 400 compounds with PKA protein spiked with both staurosporine and olomoucine, known ligands of PKA. (Left) TIC, UV trace at 214 nm, and corresponding mass chromatograms for olomoucine and staurosporine. (Right) ESI mass spectra for olomoucine and staurosporine obtained from the peaks of the mass chromatograms identifying both ligands. Reprinted from reference [16] with permission from Elsevier Science.

mass spectral and UV raw data illustrate the reliability of the GPC spin column methodology with HPLC ESI-MS and UV detection. The observed primary hits are confirmed by repeating the experiments with the single compounds in the presence and absence (control) of protein, in triplicate. In a screening campaign for ligands non-covalently bound to a ubiquitin-conjugating enzyme target (MW 25 kDa), the analysis of @500000 compounds took @1 month, @9 days for the primary screens (unattended operation) and the remainder of the time for data evaluation and the confirmation and control screens. Of the 151 compounds which were primary hits (0.03% hit rate), 23 compounds were confirmed hits (0.005% hit rate). The total amount of protein consumed was @0.5 mmol (@9 mg for the 25-kDa protein) and the cost of consumables was <$10 000. This methodology has also been applied to orphan protein targets, molecular targets with unknown function, including transcription factors, adapter molecules, regulatory subunits, heat shock proteins, metal binding proteins, RNA binding proteins, phosphatases, oxidoreductases and other proteins [16, 19, 20]. IC50 values up to the 10 mM range were detectable using the GPC spin column/ESI-MS methodology. Detection of low-affinity ligands is most likely related to compounds with low off-rates in the GPC step.

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