Preliminary validation of a computer-based approach to find new lead molecules targeting the oncogenic Forkhead box M1 transcription factor

• Author / Creator

  Elshaikh, Asya S Egweda

• The upregulation of the forkhead box M1 (FOXM1) transcription factor is directly correlated with cancer initiation, invasion, and drug resistance. Because the overexpression of FOXM1 is considered an important factor in carcinogenesis, this protein could be a relevant drug target in cancer treatment, and possibly, in cancer imaging. Literature reports describe the use of (a) siRNA, (b) proteasome inhibitors (which upregulate the expression of an endogenous negative regulator of FOXM1), and (c) other small-molecule drugs targeting FOXM1, as the three main strategies employed to decrease the transcriptional activity of FOXM1 in vivo and in vitro. Nevertheless, the first two approaches are either inconvenient because of the poor stability and the inefficient oligonucleotide intracellular delivery system (siRNA), or they potentially would exert significant side effects (proteasome inhibition). For this reason, developing new small-molecule drugs, that directly target the FOXM1 protein, represents an interesting and promising research opportunity in Medicinal Chemistry. As a part of a long-term multidisciplinary research project aimed to validate the FOXM1 protein as a drug target, we recently conducted a molecular modeling (docking) approach in which we determined the theoretical binding energies of 3,323 drugs that are (or were) approved by FDA, using the reported FOXM1-DNA binding domain. The aim of this research work was to validate a computer-based (in silico) approach by correlating the calculated binding energies of hit molecules, with their ability to interfere with the transcriptional activity of FOXM1 in vitro using breast cancer (MDA-MB-231 and MCF-7) and non-cancer (MCF-10A) cells, screening for potency and selectivity, respectively. In this regard, we carried out (A) MTT colorimetric and (B) western blot assays to evaluate the effect exerted by selected drugs (troglitazone, β-estradiol-3-benzoate gliquidone, dehydrocholic acid and metocurine, using thiostrepton as a positive control) on cell viability and protein expression. To determine the ability of the test drugs to block the binding interaction between FOXM1 and DNA, we used the electrophoretic mobility shift assay (EMSA). The MTT assay results illustrated that troglitazone, β-estradiol-3-benzoate and the control drug thiostrepton are cytostatic to MDA-MB-231 cancer cells but they are not selective as they also inhibited the proliferation of normal MCF-10A cells. However, these drugs did not inhibit MCF-7 cells proliferation. Western blotting assay results showed that troglitazone significantly inhibited FOXM1 protein expression at a concentration lower than its IC50 value, suggesting that troglitazone cytostatic effect is FOXM1-dependent. Rather than decreasing FOXM1 expression, β-estradiol-3-benzoate increased protein expression as it might act by FOXM1-independent pathways. In addition, it is a hormone-like drug and is likely to be an unsuitable scaffold to design new FOXM1 inhibitors. Gliquidone gradually inhibited FOXM1 protein expression, but this inhibitory effect did not correlate with its effects on cell viability (MDA-MB-231 cancer cells). Finally, dehydrocholic acid and metocurine did not show any effect on cell viability or protein expression, which rules them out as scaffolds to design new FOXM1 inhibitors. Regarding the EMSA assay, we could not modify this assay to work with our recombinant FOXM1 DBD-DNA complex; accordingly, we could not validate the in-silico model to determine the mechanism of drugs on cell proliferation and protein expression. In summary, we identified the drug troglitazone as the most promising lead molecule to be used in future drug design programs. On this regard, our group is currently developing troglitazone-based FOXM1 inhibitors capable of exerting binding interactions via a π-sulfur interaction involving troglitazone’s thiazolidinedione ring and a His287 residue, within the FOXM1 – DNA binding pocket. This binding interaction seems to be the main driving force guiding the inhibitory effects of several types of FOXM1 inhibitors, and it constitutes a novel mechanism of action for future FOXM1 inhibitors.

• Subjects / Keywords

  • FOXM1
  • Cancer
  • Transcription factors

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R37W67K75

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.