Inhibition of hypoxia inducible factor 1-transcription coactivator interaction by a hydrogen bond surrogate alpha-helix

Abstract

Designed ligands that inhibit hypoxia-inducible gene expression could offer new tools for genomic research and, potentially, drug discovery efforts for the treatment of neovascularization in cancers. We report a stabilized alpha-helix designed to target the binding interface between the C-terminal transactivation domain (C-TAD) of hypoxia-inducible factor 1alpha (HIF-1alpha) and cysteine-histidine rich region (CH1) of transcriptional coactivator CBP/p300. The synthetic helix disrupts the structure and function of this complex, resulting in a rapid downregulation of two hypoxia-inducible genes (VEGF and GLUT1) in cell culture.

Figure 1

(a) Transcription of hypoxia inducible genes is controlled by the interaction of DNA-bound HIF-1α/ARNT heterodimer with transcription coactivator CBP/p300. (b) Competitive inhibition of the HIF-1α C-TAD complex with CBP/p300 CH1 domain leads to downregulation of VEGF transcription. (c) α-Helices from the C-TAD_793–826 domain of HIF-1α bind to the cysteine-histidine rich 1 (CH1) region of CBP/p300. HIF-1α is shown in orange and yellow colors and CBP/p300 in green (PDB code 1L8C). (d) HBS α-helices feature a carbon-carbon bond in place of an N-terminal i and i+4 hydrogen bond.

Figure 2

(a) Circular dichroism spectra of HBS helices 1–3 and unconstrained peptide 4 in 10% TFE in phosphate buffered saline (pH 6.3). (b, c) Isothermal titration microcalorimetry analysis of HBS 2 binding to GST-p300. (b) Baseline-subtracted raw ITC data for injections of HBS 2 into a solution of the GST-p300 in Tris buffer at 25 °C. (c) Integration of titration signals (squares), caused by the binding of HBS 2 with GST-p300, fit to a single-site binding model curve (line).

Figure 3

(a) Inhibition of VEGF and glucose transporter 1 (GLUT1) gene expression with HBS helix 2 and peptide 4, after 12 h of incubation under hypoxia conditions, as measured by real time qRT-PCR in HeLa cells. *, P < 0.05, t-test. (b) Cell density and population doubling data for cultures treated with chetomin, HBS 2, and control peptide 4. 200 nM chetomin and 1 μM peptides (2 and 4) were used in the cell culture and cell viability assays. Control: cell culture medium only, vehicle: 0.1% DMSO in cell culture medium.