In this study, we used a cell-based HRE pathway assay in qHTS format to identify twelve compounds in an NTP compound library that stimulated the expression of HIF-1α and therefore appear to act as hypoxia mimetics. For three of these compounds and the positive compound CoCl2, hypoxia mimetic activities were confirmed in secondary assays of HIF-1α-dependent VEGF secretion and hypoxia-regulated promoter activity. Based on these secondary tests, we demonstrated that the hypoxia pathway responses induced by o-phenanthroline, iodochlorohydroxyquinoline, and CoSO4 (as well as the positive control CoCl2) are HIF-1α-dependent and produce hypoxia target gene promoter activation profiles similar to those induced by the standard hypoxic condition (1% O2). These results suggest that the use of a pathway assay in primary screening, in combination with focused secondary assays, can effectively identify chemical mimics of hypoxia among large collections of environmental chemicals.
As the hit rate from the primary screen was relatively low, we investigated the possibility of false negative compounds. One way to estimate the false negative rate in such an assay is to examine if any of the negative compounds are closely related structurally to any of the positive compounds (i.e., a structure-activity relationship analysis approach). This approach is not definitive, however, as compounds with similar structure do not necessarily exhibit the same activity. It is also possible that compounds that are not structurally related to any of the positive compounds were false negatives as a potential consequence of compound degradation. The only way to check for compound degradation is to analytically analyze the entire library, and reorder and retest those compounds that were identified as degraded. This approach was not practical for the purpose of this study, due to time and resource constraints, but will be evaluated in a subsequent round of testing with a new compound library. Thus, we used the former approach for investigating the possibility of false negatives. We calculated the Tanimoto scores (Randic, 1997
) between the positive compounds and all other compounds in the library. Assuming that all negative compounds that are structurally similar to one of the positive compounds should have been positive, which is a very conservative assumption, the estimated false negative rate for this assay is 1.2% if we adopt the traditional Tanimoto cutoff for similarity of 0.8, and 2.9% if we use the less conservative cutoff of 0.65.
In Supplementary Table 2
we have listed the five confirmed active compounds as an example, and their closest structural analogs as measured by Tanimoto similarity. The closest structural analogs of o
-phenanthroline are benzo(f)quinoline and quinoline with a Tanimoto score of 0.86. Both analogs were negative in the primary screen. As the activity of o
-phenanthroline might be attributed to its iron chelating ability, it is not surprising that the two structural analogs do not have the same activity because neither has the structural feature necessary for iron chelation. Similarly, the closest structural analog of CoSO4
, another iron chelator, is sodium hydrosulfate, which does not have the ability to displace iron, and therefore, would not be expected to be active in this assay. The other active compound that can act as a chelator is iodochlorohydroxyquinoline; its closest structural analog is 8-hydroxyquinoline (Tanimoto score, 0.7), which also contains the chelation feature. We did observe a weak response with 8-hydroxyquinoline; however, this compound was considered to be a Class 4 compound because its efficacy over the concentration range tested was < 10%. This low efficacy compound may have a real effect and needs to be further investigated.
7,12-DMBA is a polycyclic aromatic hydrocarbon (PAH). PAHs, as a class, tend to give positive responses in many reporter gene assays and indeed, other PAHs in the library, such as benzo(k
)fluoranthene, and dibenz(a
)anthracene, were active in the primary screen. However, these compounds are fluorescent, and as such, were suspected to be false positives in the primary screen. They were included in the follow-up study, however, where their activity was not confirmed. Another PAH in the collection, benzo(e
)pyrene, was negative in the primary screen. None of these four PAHs have substitutions on the polyaromatic ring system, whereas 7,12-DMBA has two methyl group substitutions, which may be critical for its activity in these hypoxia pathway assays. The only other PAH in the library that has methyl substitutions is 1-methylpyrene. This compound is the closest structural analog of 7,12-DMBA (Tanimoto score = 0.98; Supplementary Table 2
), but it was inactive at the concentrations tested (0.59nM to 92μM) in the primary screen.
6-Methylcoumarin is the closest structural analog of 7-DEA-4-MC (Supplementary Table 2
) but it has a low Tanimoto similarity score (0.65) and was inactive in the primary screen. The mechanism of action for 7-DEA-4-MC in this assay is not known, but if its activity was due to the coumarin scaffold, then 6-methylcoumarin is likely a false negative.
Of the twelve compounds identified as active in the primary screen, ten were confirmed in the primary assay and five showed activity in the VEGF secretion assay. Of these, three plus the positive control compound (CoCl2) showed HIF-1α dependence and an ability to mimic hypoxia in promoter activation, so these four compounds can be confidently designated as hypoxia mimics. The two other compounds, 7-DEA-4-MC and 7,12-DMBA, will require further investigation to determine why they acted differently in the secondary screens.
Based on EC50
values, there is discordance in the ranking order of compound potency between the HRE-bla
assay and the VEGF secretion assay. Although the basis for this discordance is unknown, one possibility is that it reflects differences in the HRE used to drive the β-lactamase reporter gene versus the endogenous VEGF. One of the positive compounds, cobalt (Maxwell and Salnikow, 2004
), had previously been suggested to be a hypoxia mimetic, which was confirmed in this study. It has also been shown that VEGF mRNA (Namiki et al., 1995
) and protein levels (Dai et al., 2008
) are significantly increased by CoCl2
, a finding that are consistent with hypoxia responses. We also found from our primary screening and follow-up studies that o
-phenanthroline and iodochlorohydroxyquinoline acted as chemical hypoxia mimetics, suggesting that this approach is useful for identifying previously unsuspected hypoxia mimetics.
Pathway reporter assays such as that used here are powerful tools to identify compounds acting at any one of a number of steps in a biological process leading to changes in gene expression (Xia et al., 2009
). However, they rely on relatively small synthetic response elements that may not recapitulate the context in which they function in individual genes, where they often exist in the context of larger hypoxia-induced elements, hypoxia-repressed elements, and nonresponding sequences. Thus, examination of the effect of compounds identified in such reporter screens on endogenous transcriptional regulatory elements represents a useful approach to confirming genomic relevance of the primary findings. The panel approach used here provides a level of comprehensiveness intermediate between single synthetic regulatory elements and genome-wide studies of expression or factor binding.
It has been shown that hypoxia can be induced by divalent metal ions such as cobalt and nickel (Maxwell and Salnikow, 2004
) and by iron chelators such as desferrioxamine (DFO) (Wang and Semenza, 1993
) under normoxic conditions. The likely mechanism of HIF-1 activation by these compounds is that nickel and cobalt can substitute for the ferrous ion in regulatory dioxygenases which leads to inactivation of the enzymes (Maxwell and Salnikow, 2004
). In addition to iron substitution, nickel and cobalt also bind more tightly than ferrous ions to the membrane transporter DMT-1 (divalent metal transporter 1), thereby blocking delivery of ferrous ions into cells (Maxwell and Salnikow, 2004
). In this study, we found that o
-phenanthroline, an iron chelator (Rauen et al., 2007
; Ryter et al., 2000
; Vasconcelles et al., 2001
), stimulated the HIF-1 signaling pathway and activated HIF-1α–dependent VEGF secretion. The profile of hypoxia-responsive promoter activities in the presence of o
-phenanthroline is very similar to the activity profile seen under low oxygen (1% O2
) conditions or after treatment with cobalt, which suggests that o
-phenanthroline may act in similar fashion to DFO by depriving cells of free ferrous ions which are essential for the activity of regulatory dioxygenases.
In comparison to cobalt and o
-phenanthroline, iodochlorohydroxyquinoline also stimulated the HIF-1 signaling pathway and activated HIF-1α–dependent VEGF secretion, but had a slightly weaker hypoxia-regulated promoter activity correlation with 1% O2
. These results suggest that iodochlorohydroxyquinoline may induce hypoxia though a different mechanism(s) than cobalt and o
-phenanthroline. Iodochlorohydroxyquinoline, also known as clioquinol, is a Cu(II)/Zn(II) specific chelator. Consistent with our findings, iodochlorohydroxyquinoline has been shown to increase functional HIF-1α protein, leading to increased expression of its target genes, VEGF and EPO, in SH-SY5Y and HepG2 cells (Choi et al., 2006
). Iodochlorohydroxyquinoline inhibited ubiquitination of HIF-1α in a Cu(II)- and Zn(II)-dependent manner. It prevented FIH-1 (factor inhibiting HIF-1α) from hydroxylating the asparagine residue (803) of HIF-1α, which leads to the stabilization of the trans-active form of HIF-1α (Choi et al., 2006
The recent National Research Council (2007)
report on “Toxicology in the 21st
Century” recommends a future emphasis on evaluating perturbations to “toxicity” pathways in cultured human cells. In response to this report, the NTP, the NCGC, and the U.S. Environmental Protection Agency entered into a Memorandum of Understanding in early 2008 to utilize their complementary expertise and capabilities in the research, development, validation, and translation of new and innovative test methods that characterize key steps in toxicity pathways (Collins et al., 2008
; see also http://ntp.niehs.nih.gov/go/28213
). The goal of the “Tox21” partners is to transform toxicology into a high-throughput, predictive, and mechanism-based science (Kavlock et al., 2008
), in order to evaluate the thousands of compounds to which humans are exposed and for which there is little or no toxicological information (Judson et al., 2008
). The results reported here, identifying compounds that are active in the hypoxia-response pathway, provide evidence that this pathway approach is both tractable and reliable for identifying compounds of potential toxicological interest.