Binding Affinities Towards Different STAT Proteins

4.1.1. Binding Assay

The group of T. Berg has developed a high-throughput fluorescence polarization assay which allows the screening of small molecules that can bind to the STAT SH2 domain and thereby inhibit its activity. This assay is based on the binding of small fluorescein-labeled phosphotyrosine-peptides to unphosphorylated STAT. In nature, the unphosphorylated STAT binds to kinases and is then phosphorylated by them. This tyrosine phosphorylation of STATs induces dimerisation and with that activates the STAT. If the binding of STAT to the kinase is inhibited, the activity of the STAT is blocked.

The developed polarization assay is based on this interaction. At first, an unphosphorylated STAT is incubated with a short fluorescein-labeled phospho-peptide (such as peptides P1 – P5) with a sequence derived from the active site of kinases known to interact with the STAT. A small molecule inhibitor can displace the peptide bound to STAT protein. The bound peptide exhibits a decrease in fluorescence polarization due to its significantly reduced molecular mass.¹¹⁴

4.1.2. Binding Results and Discussion

The binding affinities of receptors 66, 70, 74 and 77 – 79 towards the different peptides P1, P2 and P3 were investigated.¹

a) 5-Carboxyfluorescein–Gly-pTyr-Asp-Lys-Pro-His-Val-Leu-OH (P1) b) 5-Carboxyfluorescein–Gly-Phe-Asp-pThr-Tyr-Leu-Ile-Arg-Arg-OH (P2) c) 5-Carboxyfluorescein–Gly-pTyr-Glu-Glu-Ile-Pro-OH (P3) The complexes 66, 70, 74 and 77 – 79 are potential receptors for the peptides because all have a phosphorylated amino acid side chain (pTyr or pThr) and an aspartic or glutamic acid as a second binding motif in their sequence.

1 Peptides were synthesized by Dr. F. Freudenmann. Binding investigations were performed by Bianca Sperl under the supervision of Dr. Thorsten Berg (Max Planck Institute of Biochemistry,

4.1.2.1. Inhibitory Effect Against the P1-STAT1-Complex

The influence of the metal complexes 66, 70, 77 – 79 on the binding between P1 and STAT1 was investigated by the detection of the fluorescence polarization change of the system upon addition of the receptors. The diagram in Figure 34 shows the dependence of the peptide – protein binding on the concentration of added receptor. (The y-axis shows the ratio of P1-STAT1-complex in %, the x-axis the receptor concentration in μM) As a reference, the receptor substructure complex 80 was used for all measurements. The influence of this receptor (80) should appear at higher concentrations when compared to the other receptors as it consists of only one binding site and should therefore have a lower affinity. All measurements were conducted in duplicates under the same conditions

Figure 34: Titration curves of receptors 66, 70, 77 – 79 and the reference compound 80 in the fluorescence polarization assay with the P1-STAT1-complex.

inhibitor concentration [μM]

% binding P1-STAT1

80 77 78 79 66 70

The measurements revealed that receptors 66, 77 and 78 have an influence on the P1-STAT1 binding at a concentration of about 300 μM while slightly higher concentrations of receptors 70 and 79 are needed to show the same effect (400 μM and 500 μM, respectively). The reference compound 80 inhibits the complex formation at a concentration of nearly 1 mM. These results indicate that the structure of the complexes has little influence on their inhibitory effect. Only the two largest compounds 70 and 79 of each group are slightly less active than their smaller homologues.

The results show that all bidentate receptors have an increased inhibitory effect when compared to the monodentate substructure 80. The complexes 66, 77 and 78 show the same effect at a threefold lower concentration while the complexes 70 and 79 do so at half the inhibitory concentration of 80. This shows that the synthetic receptors with two binding sites have a higher affinity to the target peptide in the competitive binding than the one with only one binding site. However, the kind of the second binding site is not decisive, as comparable results were obtained regardless of whether the second binding site is a guanidine moiety (receptors 66 and 70) or a Zn(II)-NTA complex (receptors 77 – 79).

4.1.2.2. Inhibitory Effect Against the P2-STAT3-Complex

Additionally, titration experiments with the peptide-protein complex P2-STAT3 were performed. Nearly the same results as for the P1-STAT1 inhibition were obtained in these experiments. The bidentate receptors showed an influence on the P2-STAT3 binding at an inhibitor concentration of 300 - 400 μM while a slightly higher concentration of receptor 80 was needed to result in the same effect (600 μM).

As before, the length and also the kind of the second binding site of the receptors did not play an important role for the activity of the receptors. The bidentate receptors have an increased inhibitory effect being active at half of the inhibitory concentration when compared to the monodentate substructure 80. Interestingly, when comparing peptide P1 to peptide P2, the inhibitor concentration necessary for an influence on the P2-STAT3 binding when using receptors 66, 70, and 77 – 79 is nearly the same while for the complex 80 the concentration is reduced from about 1000 μM for the P1-STAT1-complex to 600 μM for the P2-STAT3-complex.

Figure 35: Titration curves of receptors 66, 70, 77 – 79 and the reference compound 80 in the fluorescence polarization assay with the P2-STAT3-complex.

4.1.2.3. Inhibitory Effect Against the P3-Gst-Lck-Complex

The titration experiments with the bidentate ligands 66, 70, 74 and the reference complex 80 with the P3-GST-Lck-complex were performed in analogy to the systems shown before. In contrast to peptides P1 and P2, P3 bears two carboxyl side chain functionalities in close proximity to the phosphorylated amino acid. Therefore it was expected to be a good binding partner for the receptors containing the bis(Zn(II)-cyclen) triazine complex and the guanidinium moiety. However, the measurements revealed almost no difference between the bidentate inhibitors 66, 70 and 74 and the reference complex 80. The concentrations of all complexes at 50 % of the P3-GST-Lck-complex were found to be in the range of 500 – 600 μM. This indicates that only one binding moiety of the bidentate receptors 66, 70 and 74 can interact with the P3-GST-Lck-complex. Therefore they show the same inhibitory effect as the reference compound 80.

% binding P2-STAT3

80 77 78 79 66 70

Figure 36: Titration curves of receptors 66, 70, 74 and the reference compound 80 in the fluorescence polarization assay with the P3-GST-Lck-complex.

4.1.2.4. Summary

In conclusion the results of the titration experiments showed that the bidentate receptors 66, 70 and 77 – 79 show an insignificantly higher inhibitory effect than the reference compound 80, at least for the peptide protein complexes P1-STAT1 and P2-STAT3. For the P3-GST-Lck-complex this is not the case. Further, no relationship between the structure of the receptor and the binding affinity could be determined.

Because these indirect measurements based on the influence of the metal complexes on the inhibition of the interaction between the short fluorescein-labeled peptides P1 - P3 with their natural partners (STAT1, STAT3 and GST-Lck) were not entirely successful, it was decided to determine the affinity of the metal complexes towards different peptides directly through fluorescence measurements.

80 66 70 74

inhibitor concentration [μM]

% binding P3-GST-Lck