HNF4α is an attractive target for pharmacologic manipulation. Not only is it at the center of multiple complex feedback loops that maintain differentiated function in the pancreas and liver, but HNF4α has also been implicated in a number of disease states, including diabetes, inflammatory bowel disease, cancer, and others. Here, we report the discovery of small molecule inhibitors of HNF4α that help interrogate and clarify complex processes driven by HNF4α-dependent pathways.
Until about a decade ago, HNF4α was considered to be an orphan receptor. Structural studies identified tightly bound fatty acids in the LBP of HNF4α, which existed in a mixture of active and inactive conformations, suggesting the fatty acids were playing a structural role, rather than inducing a specific conformational change (Dhe-Paganon et al., 2002
; Wisely et al., 2002
). Affinity isolation followed by mass spectrometry (AIMS) was used more recently to reveal that linoleic acid (LA, C18:2ω6) was bound to HNF4α in the livers of fed but not fasted mice, suggesting that ligand binding could be reversible (Yuan et al., 2009
). These results were also consistent with findings in Drosophila
showing that a GAL4-dHNF4 ligand sensor could be activated by starvation or administration of exogenous long chain fatty acids (Palanker et al., 2009
). However, the study by Yuan et al. did not find evidence of a significant effect of ligands on HNF4α transactivation. In contrast, we found that MCFAs and LCFAs, including the polyunsaturated fatty acid linoleic acid, antagonized the insulin promoter, while shorter chain fatty acids, which have not been found in the LBP of HNF4α (Dhe-Paganon et al., 2002
), did not. Our ability to observe effects of fatty acids on HNF4α activity in T6PNE cells may be because HNF4α expression is induced in those cells by tamoxifen simultaneously with the addition of fatty acids.
The observed correlation between fatty acids that have been found to be bound to HNF4α and those that have a repressive effect on the insulin promoter assay, together with evidence that HNF4α expression is inhibited by fatty acids, suggests that fatty acids are weak HNF4α antagonists. This is consistent with the finding that linoleic acid inhibited HNF4α protein expression (Yuan et al., 2009
). The finding that fatty acids antagonize HNF4α activity may provide insight into the mechanism by which fatty acids exert their biological effects, e.g., in βcell lipotoxicity as well as other disorders in which there are high circulating levels of fatty acids.
Based on the data presented, we believe that BIM5078 and BI6015 act as HNF4α antagonists by binding directly in the LBP. This thinking is supported by our biochemical data showing tight binding, although we can’t use those results to differentiate orthosteric from allosteric interactions. BIM5078 and BI6015 are structurally similar to FK614, a known PPARγ agonist, and we show that BIM5078 retains PPARγ agonist activity, strongly indicating that the compound binds within the PPARγ LBP. FK614 bound poorly to HNF4α and was inactive in all of the assays for effects on HNF4α activity. Of particular interest, PPARγ and HNF4α, both of which are affected by BIM5078, share fatty acids as their natural ligands. This provides support that BIM5078 and BI6015 are HNF4α ligands. Consistent with that, our docking experiment shows that BIM5078 and BI6015 have binding poses that are similar to the fatty acids observed in HNF4α crystal structures. The effect of BIM5078, acting as an antagonist of HNF4α and an agonist for PPARγ, is similar to that of fatty acids that potently activate PPARγ, while we and others found them to inhibit HNF4α (Yuan et al., 2009
). Regardless, the atomic details of BIM5078 and BI6015 interaction with HNF4α are yet to be revealed and depend on the development of ligands with improved solubility to enable future structural work.
Genetic deletion of HNF4α in pancreatic β-cells did not result in loss of insulin gene expression (Gupta et al., 2007
), in contrast to our results with T6PNE cells that displayed high sensitivity to small molecule modulation of HNF4α activity and enabled discovery of viable antagonists in the short time frame in which HTS is performed. We show that HNF4α not only appears to act on a number of target genes through effects on E47, which we induced at a submaximal level in T6PNE for the screening purposes, but it is also induced by E47 expression, a previously unknown mechanism of HNF4α transcriptional regulation. This suggests that HNF4α and E47 form a multicomponent regulatory network involving complex feedback loops, similar to what has been described for the interaction between HNF4α and HNF1α (Odom et al., 2004
). We think that it is this relationship between E47 and HNF4α that renders the insulin promoter in T6PNE cells susceptible to HNF4α modulation, affording us the opportunity to discover compounds that antagonized HNF4α activity.
The effect of HNF4α antagonists on E-box containing genes was unexpected, as the vast majority of these genes are not known to contain HNF4α binding sites and do not bind to HNF4α in ChIP-Chip assays (Odom et al., 2004
). However, a similar phenomenon was reported for the human intestinal cell line Caco-2, in which two-thirds of the genes that bound HNF4α in their ChIP-Chip assay had no discernable HNF4α binding site (Boyd et al., 2009
). Classically, NRs exert their effects by binding to highly conserved DNA binding elements, but it is known that NRs can also act indirectly via tethering to target genes (Adler et al., 1988
). NR tethering has been reported to occur through a number of DNA binding transcription factors, including members of the bHLH class including E47 (Murayama et al., 2004
) and the homeodomain class (Stender et al., 2010
). On the human insulin promoter, which does not contain an HNF4α binding site and does not bind to HNF4α in ChIP-Chip assays (Odom et al., 2004
), we found that binding of both E47 and PDX1 was inhibited by BIM5078, which we propose to be an HNF4α antagonist. On the p21 promoter, HNF4α was shown to act through a DNA binding independent mechanism involving binding to the bHLH factor c-myc (Hwang-Verslues and Sladek, 2008
). Altogether, these results suggest a model in which HNF4α is recruited to a transcriptional complex bound to DNA in a ligand-dependent manner, but might not necessarily involve direct binding of HNF4α to DNA.
While studying the effect of BIM5078 and BI6015 in vitro
, we noticed marked toxicity in a variety of tumor cell lines but not in cells cultured from primary tissue. HNF4α has previously been described to have a role in tumor pathogenesis, but the studies are conflicting, with both upregulation and downregulation of HNF4α expression being reported in association with tumor progression. Knockdown of HNF4α mRNA by siRNA has been shown to inhibit growth and proliferation of colorectal cancer cells in vitro
(Schwartz et al., 2009
). This is consistent with reports that have shown that HNF4α is upregulated in human hepatocellular carcinoma (Xu et al., 2001
). However, others have shown downregulation of HNF4α promotes tumorigenesis in hepatocellular and other cancers (Ning et al.).
Several lines of evidence suggest that the effects of BI6015 on transformed cells are mediated through HNF4α. First, we detected marked toxicity with selective HNF4α knockdown by siRNA. Furthermore, the concentration at which BI6015 induced cytotoxicity was very similar to that at which it affected the expression of downstream targets of HNF4α. This would not be expected if the effects on cancer cells were caused by an off-target effect.
, BI6015 caused dose-dependent hepatic steatosis in normal hepatocytes and in Hep3b xenografted cells. This is consistent with the effects of genetic ablation of HNF4α (Hayhurst et al., 2001
). Importantly, the induction of steatosis provided a biomarker for where the compound was acting, which appeared to be restricted to regions surrounding blood vessels. Apoptosis was induced in Hep3b cells but not in primary hepatocytes, consistent with the in vitro
results. Of note, apoptotic cells also were distributed in a perivascular pattern, mimicking that of steatosis. The lack of activity of BI6015 distal to the vessels suggests either poor tissue penetration, as has been shown for a number of chemotherapeutics including doxorubicin (Minchinton and Tannock, 2006
), or extensive hepatic metabolism that limits the amount of active compound to regions surrounding vessels. The in vitro
pharmacology with hepatic microsomes suggests that extensive hepatic metabolism is likely to be occurring in vivo
, supporting the need for additional medicinal chemistry to develop more stable molecules. We think that the limited in vivo
efficacy in the Hep3B orthostatic xenograft model could have been due to a high degree of hepatic metabolism, thereby restricting the amount of drug that penetrated into the tumor, which would be consistent with steatosis in both normal liver and tumor samples being localized to the region around blood vessels. Nonetheless, our data indicate that BIM5078 and BI6015 might be promising agents, and HNF4α a promising target for cancer therapy. In addition, these compounds provide powerful tools for studying the function of HNF4α. Additional studies will be required to determine whether an appropriate balance between the anti-tumor effects of HNF4α and the side effects of inhibiting HNF4α, e.g., hepatic steatosis, can be achieved. The fact that mice administered BI6015 for up to a month have tolerated it well raises the hope that this will be the case.