A Cyclic Peptide Inhibitor of HIF-1 Heterodimerization That Inhibits Hypoxia Signaling in Cancer Cells

Elena Miranda,Charlotte E Lawrence,Paul A Townsend,Ida K Nordgren,Francesco Cuda,Franciane Hoakwie,Ali Tavassoli,Abigail L Male,Graham K Packham,William Court,Suzanne A Eccles,Keith R Fox

doi:10.1021/ja402993u

Abstract

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that acts as the master regulator of cellular response to reduced oxygen levels, thus playing a key role in the adaptation, survival, and progression of tumors. Here we report cyclo-CLLFVY, identified from a library of 3.2 million cyclic hexapeptides using a genetically encoded high-throughput screening platform, as an inhibitor of the HIF-1α/HIF-1β protein–protein interaction in vitro and in cells. The identified compound inhibits HIF-1 dimerization and transcription activity by binding to the PAS-B domain of HIF-1α, reducing HIF-1-mediated hypoxia response signaling in a variety of cell lines, without affecting the function of the closely related HIF-2 isoform. The reported cyclic peptide demonstrates the utility of our high-throughput screening platform for the identification of protein–protein interaction inhibitors, and forms the starting point for the development of HIF-1 targeted cancer therapeutics.

Highlights

Homeostasis of oxygen, a key metabolite, is critical for mammalian cell survival
After the first round of screening, 120 surviving colonies were observed and subjected to several rounds of secondary screening to eliminate false positives and nonselective inhibitors, leaving 12 potential Hypoxia inducible factor-1 (HIF-1) dimerization inhibitors that were ranked by drop-spotting. Four of these peptides were significantly more active than the others; the SICLOPPS plasmids encoding these 4 peptides were sequenced to reveal the identity of the hypoxiainducible factor (HIF)-1 inhibitors as cyclo-CLLFVY, cyclo-CRLMVL, and cycloCLLRMY (Figure 1, R = H)
Peptides and macromolecules are increasingly viewed as the optimal scaffold for protein−protein interaction inhibitors,[40] and the high-throughout screening platform employed here has previously proven robust for the identification of cyclic peptide inhibitors of a variety of protein−protein interactions.[24,25,41]

Summary

■ INTRODUCTION

Homeostasis of oxygen, a key metabolite, is critical for mammalian cell survival. This necessitates a robust network that senses and rapidly responds to hypoxia (low oxygen levels). The reduction of luciferase signal observed in P1-treated U2OS and MCF-7 cells (Figure 2A and B) could be partially a result of a reduction in hypoxic HIF-1α levels (this would be the case for any inhibitor of HIF-1 dimerization in these cell lines). 786-O cells, a VHL-defective renal cell carcinomal line that does not express detectable levels of HIF-1α, but instead expresses HIF-2α at a high constitutive level.[35] This results in regulation of hypoxia response genes such as VEGF and lysyl oxidase (LOX) in 786-O cells by HIF-2 instead of HIF-1.36 We reasoned that if P1 inhibits the dimerization of HIF-2 in cells, a dose-dependent reduction in the mRNA and protein products of these hypoxia response genes would be observed in hypoxic 786-O cells, whereas a specific HIF-1 inhibitor would not affect hypoxia-response in this cell line. P2 and P3 had no effect on HIF-2 in this assay (Figure S9)

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES