Abstract
Oncogenic transcription factors are an increasingly important target for anticancer therapies, as inhibition could allow the “reprogramming” of tumour cells, leading to apoptosis or differentiation from the malignant phenotype. The STAT (Signal Transducer and Activator of Transcription) proteins are transcription factors that modulate a number of cellular functions. These proteins can exist as monomers, dimerise to form homodimers or form heterodimers with other family members. STAT3 induces the transcription of genes that control differentiation, inflammation, proliferation, and tumour cell invasion, and its over expression has been implicated in many tumour types. Although cancer cells are often dependent upon activation of STAT3, non tumour cells are fairly tolerant of loss of its function, most likely reflecting redundancies in normal signal transduction. Thus, STAT3 inhibitors have a high therapeutic potential. Furthermore, resistance to targeted therapies often arises from activation of an alternative signalling pathway, many of which also converge on the STATs. This suggests that inhibition of these proteins could forestall resistance in some tumour types.The STAT3 signalling pathway is stimulated by growth factors or cytokines which lead to receptor dimerization and activation. Phosphorylation of the tail of the receptor creates a docking site for the recruitment of un phosphorylated STAT3 (uSTAT3) which becomes phosphorylated at the Tyr705 position (near the C terminus) by JAK kinases. The phosphorylated STAT3 (pSTAT3) protein is then released, forming a homodimer through reciprocal binding of the SH2 domain of one monomer to the pTyr containing pYLKTKF sequence of another. This dimeric STAT3:STAT3 complex then translocates to the nucleus where it binds to its DNA consensus sequence, thus regulating transcription of numerous genes critical for the survival and proliferation of cancer cells. The protein protein interaction between two STAT3 monomers (i.e., the dimerisation event in the signalling cascade) has been identified as a valid target to inhibit DNA binding and the resultant transcriptional activation. The main objective of this research project was to synthesise novel small molecule inhibitors of the STAT3:STAT3 dimerisation process in order to identify molecules capable of down
regulating this signalling pathway as potential therapeutic agents.
A combination of advanced in silico design and pharmacophore based approaches were used to design a novel series of inhibitors capable of disrupting STAT3 dimerisation by interacting with the hexapeptide pocket of the STAT3 SH2 domain of the protein. The initial pharmacophores were taken from three ligands, RH--06 (an IL6 inhibitor), NSC--743380 and S3I--201, all previously known to have STAT3 dimerisation inhibitory properties. It was hypothesised that incorporation of these smaller fragments into one scaffold might produce molecules with a higher affinity for the SH2 domain of STAT3. The structures of these designed ligands were distinct from any previously published STAT3 inhibitors, and were found to interact efficiently with the STAT3 SH2 domain. The designed mature ligand 3.7 and the associated fragments inhibited STAT3 dimerization by 35% to 55% at 100μM which was comparable to that observed for the natural hexapeptide ligand inhibitor pYLKTKF. These compounds also had selective toxicity in the STAT3--dependent cell line MDA MB 231 with IC50 values between 6.5 μM to 10.5 μM.
Consequently, a molecular modelling directed medicinal chemistry optimisation was carried out using fragments 3.6 and 3.8, and the mature ligand 3.7 as lead scaffolds. Molecular docking was used to select amine fragments for the amide library, and boronic acid fragments for the Suzuki library. A nineteen--membered amide library (i.e., 3.8 analogues) and eleven--membered Suzuki library (i.e., 3.6 analogues) were synthesised, purified and characterised using spectroscopic techniques. The library members were screened using a Fluorescent Polarisation (FP) assay based on pure uSTAT3 as the preliminary protein--protein interaction (PPI) inhibition assay. This was followed by cytotoxicity screening against the STAT3--dependent triple negative breast cancer MDA MB 231 and the STAT3--null A4 cell line using the MTT assay. The amide library members demonstrated a wide range of STAT3 dimerisation inhibition potential and selective cytotoxicity, thus providing useful structure--activity relationship (SAR) information. In particular, compounds 4.5 and 4.13 showed notable selectivity towards the STAT3--dependent breast cancer cell line MDA MB 231, with IC50 values of 3.60 and 4.45 μM, respectively. The Suzuki library members showed only weak STAT3--dimerization inhibition potential, and had poor cytotoxicity in both the STAT3--dependent and STAT3--null cell lines.
Finally, a set of amine fragments from the amide library, including fragments present in compounds 4.5 and 4.13, along with boronic acid fragments from the Suzuki library, were selected based on their STAT3--dimerization inhibition activity and selective cytotoxicities to generate 19 mature ligands. The medicinal chemistry optimised mature ligand library members showed a notable improvement in STAT3--dimerization inhibition potency with activities ranging from 33% to 59% inhibition at 100μM, and also had sub--micromolar to low micromolar cytotoxicity in the STAT3--dependent cell line with good selectivity ratios. Compounds 6.3, 6.6, 6.10 and 6.13 were selected for molecular dynamics simulation due to their superior activity profile, and compounds 6.3, 6.4, 6.6 and 6.10 were evaluated for their ability to down--regulate STAT3--dependent genes. They were found to be able to down--regulate the STAT3--dependent genes cyclin D1, fascin and Bcl--2 in comparison to the housekeeping gene GAPDH in the STAT3--dependent breast cancer cell line MDA MB 231. The most active compound 6.6 was also evaluated in a preliminary in vivo efficacy assay in immune--compromised mice xenotransplanted with MDA MB 231 breast cancer cell line, and was found to have notable tumour growth inhibition properties.
In summary, a novel series of STAT3--dimerisation inhibitors has been synthesized, members of which possess significant protein dimerisation inhibitory activity in an in vitro Fluoresence Polarisation (FP) assay, selective cytotoxicity and the ability to down--regulate STAT3--dependent genes in a STAT3--dependent cell line, and antitumor activity in mice bearing MDA MB 231 tumours. Although further work is required to confirm the proposed mechanism of action, it may be possible to further develop this novel series of molecules in the future toward evaluation in man.
| Date of Award | 1 Jul 2017 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Khondaker Miraz Rahman (Supervisor) & David Thurston (Supervisor) |