Inhibition of FOXO3-DNA interaction by small molecules

FOXO transcription factors control apoptosis, stress resistance and longevity in mammalian cells. Although the members of the FOXO family act as tumor suppressors in some cell types, emerging evidence suggests that FOXO3 contributes to tumor stem cell renewal, immune suppression, metastasis and chemotherapy resistance. To date no compounds that directly bind and inhibit the activity of FOXO transcription factors have been described. By a combined in silico / cell biological screening approach we identified small, drug- like compounds that interact with the DNA-binding domain of FOXO3 and efficiently inhibit its transcriptional activity. To better characterize their exact mode of interaction with the FOXO3 protein, their application for cancer therapy and to generate chemical derivatives with optimized properties we will address the following tasks in this international, multidisciplinary research project: In a first step we will define the structural basis for the interaction between compound and FOXO3 by NMR spectroscopy and docking to determine the exact binding position of the substance on the surface of the FOXO3 protein. This mode of binding will provide essential information on how to perform structure-based chemical optimization of the drugs to increase their FOXO3-inhibitory potency. With these optimized drugs we will then analyze compound-dependent inhibition of target gene recognition and regulation by FOXO3 in vitro and in vivo and if these compounds can overcome FOXO3-imposed cancer cell survival and drug resistance in 3D cell culture and animals. The data on the actual mode of interaction between small drugs and the FOXO3-DNA binding domain will widen the current view on how transcription factors can be targeted with small, drug-like compounds and whether it is possible to modulate regulation of distinct target gene sets with small drugs. This study will also provide novel chemical derivatives with optimized properties regarding affinity, specificity and solubility and evaluates the relevance of FOXO3-inhibitors as therapeutic drugs for cancer types where FOXO3 promotes drug resistance and longevity of tumor cells.

The FWF-funded project "Inhibition of FOXO3-DNA interaction by small molecule inhibitors (INFODI)" identified and characterized small chemical compounds that physically interact with the DNA-binding domain (DBD) of FOXO transcription factors and thereby modulate their gene-regulatory function. FOXO3, like its family members FOXO1, FOXO4 and FOXO6, regulates a plethora of different genes, mainly acts as a homeostasis regulator and thereby controls the balance between proliferation and cell death in tissues. By pharmacophore modelling and in silico compound screening we identified candidate compounds that physically bind to the DBD of FOXO3, show selectivity among closely related FOXO family members and modulate FOXO-driven gene expression. By combining biochemical and cell-biological methods we defined the mode of binding of these substances and were the first to publish DBD-interacting FOXO-modulatory compounds worldwide. Together with our partners at the Czech Academy of Sciences and the Charles University in Prague we further developed and tested chemical derivatives of lead compounds that show biological activity in the sub-micromolar range. A publication on these derivatives is currently submitted for publication. Novel drug candidates are usually tested for efficacy, toxicity and adverse side effects in large cohorts of experimental animals. According to EU directive 2010/63 which demands a reduction and replacement of animal experimentation in research, we decided to search for alternatives in drug validation and focused onto 3D bioprinting of human tissue models. We developed a new method to directly print human tumor tissue including tumor environment into laser-manufactured fluidic chip devices and a bioink that promotes the spontaneous growth of blood vessels. This "bioprinted tumor-on-chip" model allows us to study tumor growth in a 3D human tissue context, cancer cell metastasis and tumor vascularization and to directly assess efficacy and toxicity of compounds in parallelized experiments. The model was published in the renowned journal Biofabrication, a European patent was recently filed. Although 3D bioprinted tissue equivalents do not mimic the whole complexity of human tissue, they represent a highly relevant alternative to animal experiments as they circumvent biases related to evolutionary distance between humans and mice, which are responsible for the frequently observed failure of drugs as soon as they enter clinical trials. With this, project INFODI not only developed the first FOXO-DBD targeting small chemical compounds, but also promoted the development of 3D bioprinting technology to fabricate complex human tissue surrogates for the replacement of animal experiments in drug testing and disease modelling.