Competition between Tau peptides and MT drugs: binding to tubulin

MT/tubulin targeting drugs and the crystallographic structure of their complex. The peptides used in structure determination of MT-bound Tau were allowed to compete with different compounds for binding. One compound each was selected from the classification of compounds as described before (Amos 2011) for which the binding sites are already known. The prior information obtained from the competition experiments of these compounds against hTau40 for MT/tubulin binding using ¹H-¹⁵N HSQC experiments (section 3.8.2) were expected to be complementary to the STD experiments.

3.8.3.1.1 Competition between Vinblastine (VB) and Tau peptides

The 1D STD NMR experiments were performed for all peptides used for structure determination and were compared with their 1D proton NMR spectra. It was noticed that the spectra clearly indicated the binding and specific residues involved in binding that constituted the structured region of the peptide showed highest STD effects.

STD spectra of 1mM solution of TP1, TP2, TR1, TR2, TR3 and TR’ in presence of 20µM tubulin were measured at 298K. The spectra showed clear evidence that each peptide binds to tubulin as expected. The control experiment was done using a non-binding peptide (TN1), the sequence of which constitute the N-terminal region of hTau40. No saturation transfer was observed.

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Figure 61: (a-d) Competition between Tau peptides and MT drugs for same binding site tested using STD NMR experiments. All the STD experiments were done with 1mM each of peptide and compound and 20uM of tubulin with selective saturation of the protein resonances at -0.5ppm and at 60 ppm for reference spectra. (a) The one-dimensional STD NMR spectra of the TR3 peptide in the presence of tubulin (blue) and after the addition of vinblastine (red). (b) STD NMR spectra of TR3 with tubulin (blue) and after the addition of thalidomide (red). Aromatic protons of thalidomide affected by the selective saturation of tubulin are labeled. (c) STD NMR spectra of TR3 with tubulin (blue) and after the addition of baccatin (red). (d) 1D-STD NMR spectra of TR’ peptide in the presence of tubulin(blue) and after the addition of colchicine(red) The addition of the equal concentration of the competing ligand (VB) to a sample

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containing TR3 reduced the signals of TR3 which are characteristic of the tubulin binding as shown in Figure 61a. The peaks corresponding to the side chain protons in Y310 residue at 6.7 ppm and 7.05 ppm were analyzed consistently. The resonance intensity reduced by 60% indicating strong competition between TR3 and vinblastine for the same binding site. The result is consistent with the competition of hTau40 and vinblastine for tubulin/MT binding (Figure 58a and Figure 60).

The STD based competition experiments were performed with all the peptides.

Addition of vinblastine reduced the STD intensities of TP1, TP2, TR1, TR2 and TRꞌ by 20, 50, 30, 40 and 40% , respectively. In contrast, the control peptide TN1 did not modulate binding of vinblastine to MTs, as the STD intensity of vinblastine remained unchanged by the addition of TN1 peptide.

3.8.3.1.2 Competition between Thalidomide (Thal) and Tau peptides

To give further evidence that Tau doesn’t share the binding site of thalidomide, as observed in the ¹H-¹⁵N HSQC based competition, STD experiments were performed with all the peptides bound to tubulin followed by thalidomide. The data acquired with all the peptides in the tubulin bound state underline that the binding site of Tau is different compared to that of thalidomide. The overlay of the STD spectra of thalidomide and TR3 together with tubulin and of TR3 alone with tubulin is shown in Figure 61b.

3.8.3.1.3 Competition between Baccatin (Bac) and Tau peptides

The addition of the equal concentration of the ligand baccatin to a sample containing TR3 with tubulin didn’t affect any characteristic tubulin binding resonances of TR3. The peaks corresponding to the side chain protons in Y310 residue at 6.7 ppm and 7.05 ppm were analyzed as shown in Figure 61c. The data clearly indicate the independent binding of TR3 and baccatin. Hence the data clearly show both are targeting two entirely different binding site. The result is consistent with the competition experiment of hTau40 and baccatin for MT binding (Figure 59).

3.8.3.1.4 Competition between Colchicine (Col) and Tau peptides

The STD experiments were performed with each of the binding peptides and colchicine as it was done in the case of vinblastine. There was no competition observed

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as the resonance intensities of peptides remain largely unaffected. The STD NMR spectra of tubulin-bound TR’ and TR’ and colchicine together with tubulin are overlaid and is shown in Figure 61d. The resonances of Y394HE1 at 6.7 ppm and F378HD1 at 7.15 ppm, which is one of the residues in the binding epitope, are labeled and comparison of both spectra clearly shows that there is no competition for tubulin binding.

The STD NMR based competition experiments performed are listed in the Table 12. The complete analysis of the STD spectra of all the combinations of peptides and MT drugs can be summarized as shown in the histogram Figure 62. It is evident from the graph that only vinblastine is competing with all the Tau binding peptides. Thalidomide and baccatin didn’t show competition with any of the Tau peptides at all for tubulin binding.

Figure 62: Relative effect of STD signals intensities of different Tau peptides and MT drugs. The reduction in STD NMR signal intensities after competition between peptides and compounds is plotted. -The variation in the 1D STD signal intensities of peptides in the presence of tubulin after the addition of different compounds is presented. The horizontal dotted lines on the bars show the error in comparison of signal intensities. All the STD experiments were performed at 298 K and ligand:tubulin ratio of 40:1. The relative effect of STD signals were determined by taking the intensity reduction of the signal of a residue that forms the binding epitope.

The different degrees of reduction in STD NMR signal intensities suggest different affinities for the peptides, as TR3 and TP2 bind most strongly, followed by TR2 and TR’

and relatively weaker affinities for TR1 and TP1.

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