Additional Spin Column Methods Competition Experiments of Inhibitor Mixture with Protein Target

Competition studies of ligand mixtures with a protein target can be efficiently evaluated using the GPC spin column/ESI-MS methodology [13]. The procedure involves quantitation using ESI-MS of the ligands initially present during incubation with a protein target under native conditions and after passing through a GPC spin column. This has been demonstrated for the binding competition between two ligands, a peptidic trifluormethylene ketone (TFMK) and a dibromo-quinazalone (DBQ), with CMVP. An equimolar mixture of TFMK (MW 545 Da) and DBQ (MW 489 Da) was prepared with CMVP A144L. The molar ratios for CMVP A144L to each of the inhibitors in the mixture were 1:5:5. The CMVP-inhibitor mixture was incubated for 1 h at 25 °C and the spin column eluate was analyzed by ESI-MS. The ESI mass spectrum (Fig. 2.19C) exhibited peaks for both TFMK and DBQ. For quantitation purposes, individual ESI reference spectra were obtained from mixtures prepared (without GPC spin column analysis) as 1:1 molar ratios of CMVP A144L:TFMK (Fig. 2.19A) and CMVP A144L:DBQ (Fig. 2.19B). From the integrated areas of the reference compounds (Fig. 2.19A, B) and eluted compounds (Fig. 2.19C), the molar ratios of recovered CMVP A144L:TMFK:DBQ were calculated to be 1.0:0.2:2.2. These results suggest that DBQ prevents the binding of TFMK to CMVP. It is possible that DBQ and TFMK compete for the same site and that DBQ was more strongly bound to that site than TFMK. Alternatively, the binding of DBQ to more than one site of CMVP may induce a conformational change that prevents TFMK from binding.

Fig. 2.19 GPC spin column/ESI-MS non-covalent binding competition study to determine the relative binding affinities of CMVP A144L with TFMK (MW 545 Da) and DBQ (MW 489 Da). ESI mass spectra obtained from the GPC spin column screening assay for a mixture of the inhibitors TFMK and DBQ, which form non-covalent complexes with CMVP A144L. (A) ESI mass spectrum of a reference mixture (no GPC spin column used) consisting of a 1:1 molar ratio of CMVP A144L:TFMK. (B) ESI mass spectrum of a reference mixture (no spin column used) consisting of a 1:1 molar ratio of CMVP A144L:DBQ. (C) ESI mass spectrum of a spin column eluate of a mixture of CMVP A144L:TFMK:DBQ with a recovered molar ratio of 1.0:0.2:2.2, initially prepared with molar ratios of 1:5:5, respectively. The molar ratio of CMVP A144L:TFMK:DBQ recovered after passing through the spin column was computed from the integrated areas of the peaks in Fig. 2.19A with respect to the corresponding reference peaks in Fig. 2.19B, C. Note that all spectra are normalized to the same intensity scale and were obtained using 10 mL of the mixture where the protease concentration was 20 mM in each sample. Reprinted from reference [13] with permission from John Wiley & Sons.

Competition binding experiments between mixtures of compounds and the insulin-like growth factor receptor (IGFr) protein were conducted to determine the relative binding affinities of the compounds to the IGFr protein. The IGFr protein is a potential target for inhibition by anti-cancer agents. The underlying theoretical assumptions for these competition studies are that, when the binding site and the off-rates for the drug candidates are identical, the relative ESI mass spectral responses for the drug candidates are inversely related to their Kds (EC50s) and IC50s (see Section Two mixtures of three compounds each were prepared and incubated with IGFr protein. One compound, WY360, common to both mixtures, served as a reference/calibration point for comparing the affinities for all the compounds in both mixtures. Three experiments were performed for each mixture and the ESI mass spectral data for the molecular ion regions for each of the components are illustrated in Fig. 2.20. Figure 2.20A illustrates the molecular ions generated from the GPC spin column eluate of the drug mixtures incubated with IGFr. The S/N ratio for the molecular ion of each component was computed and these are listed in Table 2.4. Figure 2.20B illustrates the molecular ions generated for each component from the GPC spin column el-uates of the mixtures without IGFr. This control experiment indicates that no compounds passed through the GPC spin columns, thereby validating the non-covalent binding results obtained when the IGFr protein was present with the compounds. Figure 2.20C illustrates the ESI mass spectral responses for each of the compounds present in the mixtures, each of equal concentration (without using GPC spin columns). The S/N ratios for each of the individual components in the mixture were determined and are listed in Table 2.4. The ratio of each ESI-MS response for the individual components for the spin column eluates when compared to that of the response factor for the pure drugs corresponds to the relative binding affinity for each of the compounds: the higher the ratio the stronger the non-covalent binding affinity and the lower the expected Kd and IC50. The response of WY360 was used to normalize the responses from both mixtures. As indicated in Table 2.4, the order of the binding affinities measured by the GPC spin column/ESI-MS method correlates with the reported IC50 values. The dynamic range of measurable binding affinities using this technique is limited by the linear dynamic range of the signal detected by the Tof mass spectrometer used (@103 counts s-1) and the concentrations of the samples used in the experiments. In all these competition experiments, the concentrations of all the compounds are identical and greater than that of the protein.

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