In the present study, we have engineered a ME180 cell line with a β-lactamase reporter under the control of a HRE that enables a reagent addition-only and microvolume (7 μL) screening assay for identifying small molecule inhibitors of the canonical HIF-1 signaling pathway. We have identified and confirmed compounds from several novel structural series that are inhibitors of the HIF-1 mediated pathway using a qHTS approach. These compounds showed good potency/efficacy, specificity against this pathway with no or low cytotoxicity, therefore might be good candidates for further testing in other cancer cell lines or animal models. The screening process and follow up studies were summarized as a flow chart in Figure .
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 (more ...)
Assays designed for the identification of modulators of the hypoxia pathway can be developed from individual pathway proteins or cell-based systems. The former biochemical assay allows identification of compounds which, for example, directly bind to the HIF-1 protein [27
], but absent the cellular context which may influence in vitro
cell activity or regulation. Therefore pathway assays can provide additional opportunities to modulate the target activity, although can require considerable follow-up studies to determine the specific site of action of the compound. However, screening a compound collection against a cell-based HIF-1 assay, such as the β-lactamase reporter gene assay, allows the identification of cell membrane permeable HIF-1 inhibitors that can be rapidly validated in secondary cell assays such as the hypoxia-induced HIF-1α accumulation and VEGF secretion assays used in this study.
The use of β-lactamase as a reporter with the cell-permeable substrate, CCF4, permits the measurement of the hypoxia response in live cells. The ratiometric readout by using this β-lactamase substrate and dual emissions (460 and 530 nm) minimizes the well-to-well and plate-to-plate variations caused by subtle differences in cell numbers that can result in the automated dispensing step. The green fluorescence signal (530 nm) is reflective of the cellular uptake of CCF4-AM substrate and can be used as an indication of cell viability. Therefore, it can be used to monitor compound cytotoxicity, independently from the response due to β-lactamase inhibition reflective of the pathway activity. For example, chetomin, a known HIF-1 inhibitor [28
] with an IC50
value of 37.5 nM in this assay, was found to cause cytotoxicity at a concentration >200 nM under the induction of HIF-1 activity via CoCl2
]. The green fluorescence decreased as well as the blue fluorescence (460 nm) for this compound, indicating that chetomin inhibited CoCl2
induced HIF-1 activity and resulted in cytotoxicity at high concentrations.
As the cell-based HIF-1 assay, conducted in the presence of CoCl2, involves the activation of a β-lactamase reporter, it may identify a number of non-specific inhibitors that appear as positive either by competing with intracellular iron or inhibiting mRNA transcription, protein synthesis or β-lactamase activity. We have applied several follow-up assays including functional assays that measure hypoxia-induced HIF-1α accumulation and VEGF secretion, an HRE-bla assay conducted in a hypoxia chamber (≤1% oxygen), which serves as a confirmation assay of the CoCl2-induced HRE-bla assay, and an assay that detects compound cytotoxicity by measuring intracellular ATP content. In order to determine the specificity of the HIF-1 inhibitors identified from the primary screen and follow-up study, we also examined their activity in other signaling pathway assays run at the NCGC. The qHTS approach combined with several follow-up studies allowed us to efficiently identify the compounds capable of inhibiting the HIF-1 signaling pathway. Of the ten structural series and 6 singletons initially identified from qHTS, NCGC00043898, NCGC00044926, NCGC00049606 and NCGC00056044 had their activity confirmed in all follow-up studies, displaying potencies ranging from 0.5-3 μM. These compounds have not been reported previously as inhibitors of the HIF-1 signaling pathway and appeared to be selective toward this pathway as compared to the NFκB and AP-1 signaling pathways. In addition, we have demonstrated that these compounds blocked VEGF secretion, a downstream response to activated HIF-1.
Using the qHTS approach potency and efficacy of each compound can be obtained rapidly from the primary screening data, limiting the amount of follow-up compound acquisition and re-testing required. Confirmation rates are greatly improved in qHTS compared to conventional single concentration screening. For example, in our follow-up studies 36 of 40 compounds re-tested positive, a confirmation rate of 90%. We also confirmed thirteen of the thirteen compounds in the NCI diversity collection identified previously by two independent laboratories [23
] (Table S3, Additional file 4
), although the methods of hypoxia induction were different among all three labs. Of these compounds, NSC-259968 and NSC-259969 showed a potent inhibitory effect on the HIF-1 signaling pathway with IC50
s of 11.3 and 10.9 nM. Two other compounds that were active are the DNA topoisomerase I inhibitors, NSC-606985 (camptothecin) and NSC-609699 (topotecan) with IC50
s of 69 nM and 240 nM, respectively. Chau et al [23
] excluded NSC-606985 and NSC-609699 as potential inhibitors of HIF-1 signaling due to their unacceptable cytotoxicity. We found that all four compounds not only displayed cytotoxicity as shown by a decrease in the green fluorescence signal (530 nm), but were also nonselective, as they blocked other pathways such as the AP-1 and NF-κ-B pathways as well as the HIF-1 signaling pathway (data not shown). The cytotoxicity of these compounds could not be detected in the HRE-luciferase reporter gene assay where they were identified as active because both cytotoxicity and inhibition of HIF-1 pathway would both result in a decrease in signal, thus the two modes are indistinguishable in this assay format.