2. Receptor Development
2.1. Potential Targets
We chose peptide sequences resembling the C-terminal domain of RNA polymerase II and the signal transducers and activators of transcription for our ditopic receptors because of their great biological relevance.
2.1.1. The C-Terminal Domain of RNA Polymerase II
The synthesis of mRNA by the RNA polymerase II (Pol II) requires precise regulation of the different transcription steps as there are the initiation, elongation and termination processes. The transcription elongation process is affected in part by the binding of regulatory factors to the phosphorylated C-terminal domain (CTD) of the Pol II.49 The binding of these factors depends on a specific CTD phosphorylation pattern, which changes during the transcription cycle, due to the action of CTD-modifying enzymes and coordinates events of nuclear mRNA biogenesis.50
The CTD is linked to the Pol II via a flexible linker and forms an extension from the Pol II.
Due to the high mobility of the linker and the CTD, they are not visible in the crystal structures of yeast Pol II.51 The CTD sequence consists of heptapeptide repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats depends on the species and is 26 in yeast and 52 in human. At least eight repeats are required for yeast viability.
52, 53
Figure 11: Pol II crystal structure and CTD β-spiral model. (adapted from literature54)
The CTD can be modified at five of the seven side chains of one consensus repeat.
However, CTD phosphorylation occurs mainly at Ser2 and Ser5 and these two phosphorylations are not equivalent in function.55, 56
H3N H N O
O
OH HO
N N
H
O H
N O HO O
N
O NH O OH O
Tyr1 OH
Ser2
Pro3 Thr4
Ser5
Pro6 Ser7
Figure 12: Structure of the diphosphorylated heptapeptide in complex.
Phosphorylation at Ser2 and Ser5 results in four different phosphorylation states of one CTD repeat. The phosphorylated form of Pol II carries, on average, one phosphate per repeat.57 The exact phosphorylation state results from the balanced action of CTD kinases on the one side and phosphatases on the other side. During the transcription cycle the phosphorylation pattern changes, resulting in activation of specific RNA-processing factors. For example the CTD becomes phosphorylated at Ser5 during transcription initiation, while the adjacent transcription elongation is triggered by CTD phosphorylation at Ser2 residues.58 Thus, it seems that the different CTD phosphorylation patterns connect distinct stages of the transcription cycle to the associated RNA-processing events.59 During or after transcription termination, the CTD is dephosphorylated, resulting in Pol II recycling.
2.1.2. The Signal Transducers and Activators of Transcription
Other targets of interest were the signal transducers and activators of transcription (STATs). This family of transcription factors transduce signals from the surface of a cell to the nucleus.60 Up to now, seven members of the STAT family were identified. STATs bind to activated cytokine receptors or to growth factor receptors via their Src homology 2 (SH2) domain. Upon ligand-induced receptor dimerisation, receptor-associated Janus kinases (JAKs) phosphorylate the cytokine receptors to create binding sites for the STATs. The STATs are phosphorylated after binding by JAKs or other cytoplasmic tyrosine kinases. This phosphorylation of STATs induces dimerisation by reciprocal phosphotyrosine-SH2 domain interactions. The so formed STAT dimers are then able to
expression.61 Due to the fact that the intracellular location of STATs depends on their activation state, they are often referred to as “latent cytoplasmic” transcription factors (see Figure 13).62
Figure 13: Simplified model of signal transduction via STATs. (adapted from literature63) One member of the STAT family, STAT3, has proven to be overactive in numerous of primary human tumors and also in tumor-derived cell lines.64 Inhibition of constitutively active STAT3 results in growth inhibition and apoptosis of the tumor cell lines.65 In
proteins takes place (see Figure 13). Therefore, small molecules like the complexes shown in this work could potentially be used as inhibitors for the STAT activation.
2.1.3. Target Peptide Sequences
After the STAT and the CTD proteins were chosen as the potential targets for the ditopic receptors, in the next step some target peptide sequences were chosen as starting point for the receptor development.
In the following some short peptide sequences derived from the CTD and STAT proteins are presented which were selected as potential targets for the receptors. All of the peptides have at least two amino acids with functional groups which could serve as binding partners for the different receptor moieties presented in the following Chapter 2.2.
All peptides have a phosphorylated tyrosine, threonine or serine and in addition a second function like a glutamic acid, aspartic acid, histidine or a second phosphorylated amino acid. They were derived either from the active centers of kinases known to be involved in the STAT phosphorylation process (P1 – P5) or from the CTD sequence (P6).
a) Carboxyfluorescein–Gly-pTyr-Asp-Lys-Pro-His-Val-Leu-OH (P1) b) 5-Carboxyfluorescein–Gly-Phe-Asp-pThr-Tyr-Leu-Ile-Arg-Arg-OH 5- (P2) c) 5-Carboxyfluorescein–Gly-pTyr-Glu-Glu-Ile-Pro-OH (P3) d) 5-Carboxyfluorescein–Gly-pTyr-Leu-Val-Leu-Asp-Lys-Trp-OH (P4) e) 5-Carboxyfluorescein–Gly-pTyr-Leu-Pro-Gln-Thr-Val-NH2 (P5) f) H-Tyr-pSer-Pro-Thr-pSer-Pro-Ser-OH (P6) The red marked phosphorylated tyrosine, threonine or serine residues can be bound by the bis(Zn(II)-cyclen) complex. As a secondary binding site, histidine (marked in green) in the sequence of P1 could be coordinated by the bis(Zn(II)-cyclen) complex or the blue marked glutamic or aspartic acids (sequences P1 - P5 which might be bound by a guanidinium moiety or a Zn(II)-NTA complex are possible targets.
In addition, peptides P1 - P5 are labeled with the fluorescent dye 5-carboxyfluorescein, thus making it possible to use fluorescence intensity or fluorescence polarization spectroscopy as method for the determination of the binding affinities.