Experimental Results

The ALIS-based off-rate measurement method was applied to a proprietary series of Zap-70 Kinase inhibitors. First, an ACE50 experiment was conducted to demonstrate that the compounds bind the same site as the quench reagent staurospor-ine. As shown in Fig. 3.15, sigmoidal plots indicate that, with the exception of one compound, the ACE50 values were all very similar to one-another. Linear ratio plots of the same ACE50 data confirm that the compounds all bind isosteri-cally with respect to the quench reagent, a necessary prerequisite for effective competition.

The mixture of these compounds at 0.5 mM per component was equilibrated with a 5 mM concentration of the protein target, then the reaction was quenched with excess staurosporine (100 mM) and analyzed using ALIS every 7 min. The measured protein-ligand complex MS responses were normalized and fit to the exponential decay function above, as shown in Fig. 3.16. The raw data fit the ex-

Fig. 3.15 (A) An ALIS competition experiment with a proprietary series of Zap-70 kinase inhibitors at 0.5 mM per component plus receptor at 5 mM concentration yields similar ACE50 values, indicating that all but one of the compounds have similar Kds. (B) Linear ratio plots of the ACE50 data in (A) confirm that the compounds all bind isosterically with respect to staurosporine.

Fig. 3.15 (A) An ALIS competition experiment with a proprietary series of Zap-70 kinase inhibitors at 0.5 mM per component plus receptor at 5 mM concentration yields similar ACE50 values, indicating that all but one of the compounds have similar Kds. (B) Linear ratio plots of the ACE50 data in (A) confirm that the compounds all bind isosterically with respect to staurosporine.

ponential function well; the fit for one of the compounds shown as an example. Varying rates of dissociation were observed for the mixture components, which was surprising given the very similar ACE50 values mentioned above, and similar IC50 values determined from independent biochemical measurements (Table 3.2) [66]. This result emphasizes the importance of having orthogonal methods to assess protein-ligand interactions when evaluating and prioritizing compounds for lead discovery: Despite comparable protein-ligand binding affinities, the off-rates for these compounds vary over an order of magnitude. This aspect these compounds' binding properties could have considerable effects on other aspects of

Fig. 3.16 The ALIS-MS responses from a dissociation rate experiment for a mixture of Zap-70 ligands using staurosporine as the quench reagent. See text for details. (A) The raw data and its fit curve for NGD-6367, one of the compounds in the mixture. (B) The exponential decay curves fit to normalized

Fig. 3.16 The ALIS-MS responses from a dissociation rate experiment for a mixture of Zap-70 ligands using staurosporine as the quench reagent. See text for details. (A) The raw data and its fit curve for NGD-6367, one of the compounds in the mixture. (B) The exponential decay curves fit to normalized

ALIS-MS response data. Each curve (left to right) corresponds to the compounds listed in Table 3.1 (top to bottom). NGD-6367, from (A), is shown as a dashed line. For clarity, the raw data points are not shown in

Table 3.2 IC5o values and protein-ligand dissociation half-lives for a proprietary series of Zap-70 Kinase inhibitors.

Entry Compound ID IC50, nM ti/2, min

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