GPC Spin ColumnEsimsnmr

The coupling of the GPC spin column/ESI-MS screening results with NMR (2D 1H-15N HSQC) is a powerful method for confirming that the non-covalent binders identified by the MS experiments truly bind at the predicted active site by observing NMR chemical shift perturbations in the vicinity of the protein active site [1, 15]. In contrast, the absence of chemical shift perturbations or a random distribution of chemical shift changes on the protein surface would imply a lack of an interaction of the compound with the protein or potentially the existence of non-specific binding. The development of the GPC spin column/MS/NMR assay

Fig. 2.21 ESI mass spectra obtained from the GPC spin column/ESI-MS screening assay for a variety of CMVP mutants incubated with TFMK illustrating specificity of the protease-inhibitor complex. (A) ESI mass spectrum of inhibitor TFMK (MW 545 Da), no GPC spin column used. The TFMK response corresponds to that of the molar concentrations of protein used in panels B, C and D. (B) ESI mass spectrum of the GPC spin column eluate of CMVP A144L and TFMK, originally incubated at a molar ratio of 1:40. The measured [CMVP A1444L]/[TFMK] molar ratio for the eluate is @1:1. (C) ESI mass spectrum of the spin column eluate of CMVP S132A and TFMK, originally incubated at a molar ratio of 1:40. TFMK does not coelute. (D) ESI mass spectrum of the GPC spin column eluate of CMVP E122V/A144G and

TFMK, originally incubated at molar ratio of 1:40. The measured [CMVP E122V/A144G]/ [TFMK] molar ratio for the eluate is 1:<0.05. The mass range illustrated only covers the inhibitor region and not the higher mass range for CMVP. The peaks labeled with stars (*) at m/z 538 and m/z 598 are background peaks produced from the solvent (3% acetic acid in 1:1 water:acetonitrile, v:v). Note that all spectra are normalized to the same intensity scale. (Bottom) Scheme illustrating the stabilized (reversible) hemiacetal CMVP-inhibitor complex proposed between the triad of amino acid residues S132, H157 and H63 and TFMK. The wavy lines (~~~~~) represent CMVP with the specific amino acid residues shown. Reprinted from reference [13] with permission from John Wiley & Sons.

Fig. 2.22 ESI mass spectra acquired from the GPC spin column eluates of five consecutive library mixtures, each often components, incubated with MMP-1 to screen for compounds non-covalently bound to the protein. (A), (B) and (D), (E) ESI mass spectra for mixtures F2, G2 and A3, B3, respectively, serve as background spectra for the ESI spectrum of the sample of interest

Fig. 2.22 ESI mass spectra acquired from the GPC spin column eluates of five consecutive library mixtures, each often components, incubated with MMP-1 to screen for compounds non-covalently bound to the protein. (A), (B) and (D), (E) ESI mass spectra for mixtures F2, G2 and A3, B3, respectively, serve as background spectra for the ESI spectrum of the sample of interest

(C) mixture H2. Nearly all the ions are from the protein, buffer and solvent background except for the ions at m/z 145.8 and m/z 155.7. These two ions are protonated molecular ions for compounds with MWs of 145 Da and 155 Da that bind to MMP-1. Reprinted from reference [1] with permission from Elsevier Science.

Fig. 2.23 (A) ESI total ion chromatogram from mixture H2 (see Fig. 2.22C). Mass chromatograms for the components with (B) m/z 145.8 and (C) m/z 155.7 from the GPC spin column eluate originating from mixture H2 often components incubated with MMP-1 (the ESI mass spectrum for this mixture is illustrated as Fig. 2.22C). The evolution of these peaks with time (scan numbers) demonstrates that these are unique components that eluted from the mixture while the other eight components were retained by the GPC spin column and not observed in the ESI mass spectrum. Reprinted from reference [1] with permission from Elsevier Science.

Fig. 2.23 (A) ESI total ion chromatogram from mixture H2 (see Fig. 2.22C). Mass chromatograms for the components with (B) m/z 145.8 and (C) m/z 155.7 from the GPC spin column eluate originating from mixture H2 often components incubated with MMP-1 (the ESI mass spectrum for this mixture is illustrated as Fig. 2.22C). The evolution of these peaks with time (scan numbers) demonstrates that these are unique components that eluted from the mixture while the other eight components were retained by the GPC spin column and not observed in the ESI mass spectrum. Reprinted from reference [1] with permission from Elsevier Science.

utilized MMP-1 (collagenase), a matrix metalloproteinase and a small compound library.

To demonstrate the screening of compounds for a potential anticancer program, a small chemical library was selected and mixtures of ten chemically compatible compounds were prepared where each compound had a different molecular weight. The mixtures were incubated with MMP-1 and analyzed using the GPC spin column/ESI-MS flow injection approach. The resulting mass spectral data were analyzed in two dimensions. The ESI mass spectra of the mixtures analyzed before and after the mixture of interest were compared to identify background peaks and new peaks associated with the mixture of interest. The second dimension analyzed was the evolution in time of the mass chromatograms for each of the components of the mixture to verify that they were components of the mixture and not from the instrumental chemical background that remained

Fig. 2.24 Screening results of GPC spin column/MS/NMR assay of MMP-1 protein with library mixture H2 (see Figs. 2.22C, 2.23), illustrating the complementary nature of the MS and NMR experiments in the MS/ NMR MMP-1 assay. (Top) ESI mass spectrum of the GPC spin column eluate of the library mixture H2 shows the presence of molecular ions for two compounds (MWs 145 Da, 155 Da) indicating non-covalent binding to the MMP-1 protein. (Bottom) Expanded 2D 1 H-15N HSQC NMR spectral regions of the MMP-1 protein in the presence and absence of each of the two compounds (overlayed blue and white spectra, respectively) indicating that the p-phenyl pyridine (MW 155 Da) induces a chemical shift change for V115 in the MMP-1 active site while the 8-hydroxyquinoline (MW 145 Da) does not induce a chemical shift change at V115. Therefore, the p-phenyl pyridine binds non-covalently to the active site in MMP-1 while 8-hydroxyquinoline binds non-specifically to MMP-1. Reprinted from reference [1] with permission from Elsevier Science.

Fig. 2.24 Screening results of GPC spin column/MS/NMR assay of MMP-1 protein with library mixture H2 (see Figs. 2.22C, 2.23), illustrating the complementary nature of the MS and NMR experiments in the MS/ NMR MMP-1 assay. (Top) ESI mass spectrum of the GPC spin column eluate of the library mixture H2 shows the presence of molecular ions for two compounds (MWs 145 Da, 155 Da) indicating non-covalent binding to the MMP-1 protein. (Bottom) Expanded 2D 1 H-15N HSQC NMR spectral regions of the MMP-1 protein in the presence and absence of each of the two compounds (overlayed blue and white spectra, respectively) indicating that the p-phenyl pyridine (MW 155 Da) induces a chemical shift change for V115 in the MMP-1 active site while the 8-hydroxyquinoline (MW 145 Da) does not induce a chemical shift change at V115. Therefore, the p-phenyl pyridine binds non-covalently to the active site in MMP-1 while 8-hydroxyquinoline binds non-specifically to MMP-1. Reprinted from reference [1] with permission from Elsevier Science.

constant with time. Figure 2.22 illustrates a total of five mass spectra sequentially acquired, two from mixtures immediately prior to the mixture of interest (Fig. 2.22A, B), the mixture of interest (Fig. 2.22C) and two from mixtures immediately following the mixture of interest (Fig. 2.22D, E). Note that most of the mass spectral peaks in the mixture of interest (Fig. 2.22C) are present in most of the other spectra except for peaks at m/z 145.8 and m/z 155.7. Figure 2.23 illustrates the total ion chromatogram for the mixture of interest (Fig. 2.23A) and the mass chromatograms for the ions with m/z 145.8 (Fig. 2.23B) and m/z 155.7 (Fig. 2.23C). Note that, in both cases, mass chromatographic peaks evolve in time, confirming the fact that these two components with MWs 145 Da and 155 Da, respectively, passed through the GPC spin column non-covalently bound to MMP-1 and did not originate from the instrumental chemical background. These two compounds when analyzed in the same manner but in the absence of MMP-1 by GPC spin column/ESI-MS did not produce any detectable mass spectral peaks, further verifying that the compounds were non-covalently bound to MMP-1.

The two compounds with MWs 145 Da and 155 Da identified by GPC spin column/ESI-MS as non-covalent binders to MMP-1 were then each analyzed in the presence of MMP-1 by 2D 1H-15N HSQC NMR (Fig. 2.24). Chemical shift perturbations associated with the active site amide amino acid residue V115 was not observed for the MW 145 Da compound but was observed for the MW 155 Da compound. The NMR data verifies the fact that the MW 155 Da compound, corresponding to p-phenyl pyridine, was specifically bound to the MMP-1 active site while the MW 145 Da compound, corresponding to 8-hydroxyquinoline, was non-specifically bound to MMP-1. The p-phenyl pyridine compound was independently found by other NMR studies to bind to stromelysin (MMP-3) [30]. Limitations associated with the 2D 1H-15N HSQC NMR methodology are the need for 15N-enriched protein and the requirements of large quantities of protein due to the low sensitivity of the NMR experiments.

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