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Chemical Modulation of the Forkhead Box M1 Transcription Factor
- Author / Creator
- Tabatabaei Dakhili, Seyed Amirhossein
Thousands of transcription factors control the process of gene expression and protein synthesis in the human body. These nuclear proteins are fundamental for healthy organ development, cellular function, immunity and body response to diseases and stress. Mutated, overexpressed, or dysregulated transcription factors are linked to cancer, neurological disorders, metabolic, and autoimmunity diseases. Forkhead Box M1 (FOXM1) is a transcription factor required for the normal progression of the cell cycle as well as tissue regeneration, homeostasis, and DNA repair. However, in many types of cancer, its expression is dysregulated. As the “master regulator of the cell cycle”, its overexpression leads to tumorigenesis, angiogenesis, metastasis and poor patient prognosis. Accumulating evidence suggests that inhibition of FOXM1 as the Achilles heel of cancer could be the master key for the cancer-treatment lock. However, direct targeting of the FOXM1 transcription factor is a challenging task due to the lack of binding pocket and ligand-binding site.
In the past decade, several small molecules have been introduced with the ability to decrease the FOXM1 expression with a different and distinct mechanism. Most-recently it is found that compounds thiostrepton (previously found to inhibit FOXM1 indirectly through proteasome inhibition) and Forkhead Domain Inhibitor-6 (FDI-6) can directly bind to the FOXM1 DNA binding domain (DBD) and suppress its activity. However, the exact mechanism of binding of these agents to the FOXM1-DBD is not reported. Since the fundamental of designing inhibitors for any target is an extensive structural analysis of the target, we used a series of molecular dynamics (MD) simulations to understand the mechanism of FOXM1-DBD/DNA recognition. Next, using different approaches, we identified a binding pocket on the DNA recognition helix of FOXM1-DBD.
Furthermore, using molecular modeling and docking techniques, we found a mutual binding mode for known FOXM1 inhibitors, thiostrepton, FDI-6, and troglitazone. We found that these compounds form a complex with the FOXM1-DBD mainly through a strong pi-sulfur interaction with His287 of FOXM1-DBD. Additionally, we reported that FDI-6 forms an additional strong halogen bonding with Arg297 by its 4-fluorophenyl ring.
To provide evidence regarding the existence of halogen bonding, we chemically modified the structure of FDI-6. Then, using protein immunoblot and EMSA of recombinant human FOXM1-DBD, we proved that only those derivatives carrying halogen atoms at positions 3 and 4 are active. To validate the hypothesis of pi-sulfur interaction, we used two different pathways, chemical and biological. First, we exchanged the sulfur atom in the structure of FDI-6 and confirmed that the sulfur atom is essential for its FOXM1 inhibitory activity. Next, using site-directed mutagenesis, we mutated the His287 of recombinant FOXM1-DBD to a simple and non-aromatic amino acid (alanine) and also to an aromatic amino acid (phenylalanine). Using EMSA, we confirmed that FDI-6 was unable to bind to the alanine mutated FOXM1-DBD, while its binding was unaffected by phenylalanine mutation. These results point to the probability that the theoretical binding site we identified using molecular modeling of the FOXM1-DBD is valid.
Based on the gained knowledge of binding sites as well as the structural requirements necessary for a small molecule to inhibit this oncogenic transcription factor, we designed a series of compounds based on the structure of thiazolidinediones (troglitazone) capable of suppressing the FOXM1 transcriptional program. Among them, compound TFI-10 was able to target and decrease the cellular level of FOXM1 without affecting FOXM1’s closely related family members and key tumor suppressors, FOXO1, and FOXO3a.
We also showed that the currently known selective FOXO1 inhibitor, AS1842856, could also target FOXM1 and decrease its protein level as well as its target genes by a mechanism other than direct binding to the DBD. We demonstrated that FOXM1b could promote the expression of FOXO1 while the other FOXM1 isoform, FOXM1c, negatively regulates the level of FOXO1. We also presented using colony formation assay and gene silencing techniques that dual inhibition of FOXM1 and FOXO1 can dramatically decrease the ability of breast cancer cells to proliferate and form colonies.
- Graduation date
- Spring 2020
- Type of Item
- Doctor of Philosophy
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