Identification of small molecule compounds that inhibit the HIF-1 signaling pathway

Abstract

Background: Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1alpha and constitutively-expressed HIF-1beta. During hypoxic conditions, HIF-1alpha heterodimerizes with HIF-1beta and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1alpha protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1alpha. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.

Results: The assay is based upon a beta-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE beta-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1alpha accumulation by western blot analysis.

Conclusion: The use of beta-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.

Figure 1

A. HRE-bla ME-180 cells express β-lactamase in response to hypoxia. Cells were incubated with indicated amount of DFO or CoCl₂ for 17 hrs before β-lactamase assay was performed. B. HIF1α, but not HIF2 α, mediates the expression of β-lactamase in response to hypoxia. Cells were incubated with indicated Stealth RNAi™ duplexes for 55 hrs, and then treated with 125 μM CoCl₂ for 17 hrs before β-lactamase assay was performed. C. Western blot analysis for DFO-induced HIFα accumulation and RNAi knockdown. Cells were incubated with indicated Stealth RNAi™ duplexes for 55 hrs, and then treated with 250 μM DFO for 5 hrs before HIF1α western blot was performed as described in Materials and Methods.

Figure 2

A. Intra-plate concentration response titration curves for NSC 607097 in the screening. Each curve was measured in duplicate in column 1 of each assay plate. B. qHTS concentration response data binned into curve classes 1-3 (Class 1 curves, blue; Class 2 curves, orange; Class 3 curves, green). C. Concentration response curves for compounds in series 39 (Class 1.1 curves, blue; Class 1.2 curves, orange; Class 2.1 curves, green; Class 3 curves, light gray; Class 4 curves, dark gray).

Figure 3

Chemical structures of inhibitors for HIF-1 signaling pathway. A. Four representative inhibitors from the qHTS. NCGC00044926 is a representative from series 31, NCGC00043898 and NCGC00049606 from series 39 and NCGC00056044 from series 71. B. Number of active and inactive compounds containing the 5-thiophen-1,2,4-oxadiazole core versus the 1,2,4-oxadiazole core. The thiophen-oxadiazole core appears to be important for compound activity. Right, Leadscope Maximal Common Substructures (MCS) used to populate series 31 and 39.

Figure 4

Effect of four novel inhibitors on HIF-1 signaling pathway. Inhibition of HIF-1 mediated β-lactamase activity in HRE-bla ME-180 cells by various concentrations of NCGC00043898, NCGC00044926, NCGC00049606 and NCGC00056044 in the presence of 1% oxygen (A) and 60 μM CoCl₂ (B). The data are displayed as the percentage of control. Representative values are from a single experiment performed in duplicate. Data are expressed as mean ± SD.

Figure 5

Inhibition of hypoxia-induced VEGF secretion. ME-180 cells were pre-treated with NCGC00043898, NCGC00044926, NCGC00049606 and NCGC00056044 at various concentrations for 5 min and then exposed to 15 μM iodochorohydrozyquinoline for 17 hrs. Then VEGF level was determined by human VEGF ELISA kit. Representative values are from a single experiment performed in duplicate. Data are expressed as mean ± SD.

Figure 6

A flowchart of identification of HIF-1 inhibitors. * 350 inhibitory compounds were identified from primary screening, in which 18 structure series were generated by using Leadscope fingerprints. ** A set of criteria (Table S4, Additional file 5) were applied to exclude series containing potentially fluorescent compounds and series with low potencies or efficacies, resulting in 10 prioritized series and 6 singletons (Table S1, Additional file 1).