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Inhibition of Hypoxia-Inducible Transcription Factor (HIF-1α) Signaling with Sulfonyl-γ-AApeptide Helices - PubMed

  • ️Sun Jan 01 2023

. 2023 Sep 13;145(36):20009-20020.

doi: 10.1021/jacs.3c06694. Epub 2023 Sep 4.

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Inhibition of Hypoxia-Inducible Transcription Factor (HIF-1α) Signaling with Sulfonyl-γ-AApeptide Helices

Wei Jiang et al. J Am Chem Soc. 2023.

Abstract

The development of inhibitors that selectively block protein-protein interactions (PPIs) is crucial for chemical biology, medicinal chemistry, and biomedical sciences. Herein, we reported the design, synthesis, and investigation of sulfonyl-γ-AApeptide as an alternative strategy of canonical peptide-based inhibitors to disrupt hypoxia-inducible factor 1α (HIF-1α) and p300 PPI by mimicking the helical domain of HIF-1α involved in the binding to p300. The designed molecules recognized the p300 protein with high affinity and potently inhibited the hypoxia-inducible signaling pathway. Gene expression profiling supported the idea that the lead molecules selectively inhibited hypoxia-inducible genes involved in the signaling cascade. Our studies also demonstrated that both helical faces consisting of either chiral side chains or achiral sulfonyl side chains of sulfonyl-γ-AApeptides could be adopted for mimicry of the α-helix engaging in PPIs. Furthermore, these sulfonyl-γ-AApeptides were cell-permeable and exhibited favorable stability and pharmacokinetic profiles. Our results could inspire the design of helical sulfonyl-γ-AApeptides as a general strategy to mimic the protein helical domain and modulate many other PPIs.

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Figures

Figure 1.
Figure 1.

(A) HIF-1α/p300-mediated transcription of hypoxia-inducible genes. (B) Structure of HIF-1α/p300 complex (PDB: 1L3E) and sequence of HIF-1α CTAD (776–826).

Figure 2.
Figure 2.

Structure of the sulfonyl-γ-AA peptide. (A) Chemical structure of the sulfonyl-γ-AA peptide; (B,C) Crystal structure of sulfonyl-γ-AApeptide: (B) top view, (C) side view. (D) Schematic representation of side-chains distribution in the sulfonyl-γ-AApeptide helix scaffold. Adapted from ref 54. Copyright 2022 American Chemical Society.

Figure 3.
Figure 3.

(A) HIF-1α-helix C bound to p300, PDB code: 1l3e. (B) Structure of HIF-1α-helix C (green, key binding residues highlighted in red). (C) Helix wheel representation of HIF-1α-helix C. (D) Helix wheel representation of sulfonyl-γ-AA-peptide with sulfonyl chains designed to mimic key residues in helix C denoted with a red star. (E) A sulfonyl-γ-AA-peptide HC11 (purple) was designed to mimic helix C. (F) Overlay of HC11 with helix C. (G) Helix wheel representation of sulfonyl-γ-AA-peptide with chiral chains designed to mimic key residues in helix C denoted with a red star. (H) Sulfonyl-γ-AA-peptide HC13 (pink) designed to mimic helix C. (I) Overlay of HC13 with helix C. The helical face employed for mimicry of critical side chains in helix C is depicted with a red star in (D) and (G).

Figure 4.
Figure 4.

(A) CD spectra of sulfonyl-γ-AApeptide mimics of HIF-1α-helix C measured at 100 μM and room temperature in PBS buffer. (B-G) Fluorescence polarization binding assay of FITC-labeled HIF-1α mimics with the CH1 region (323–423) of p300. (H) Kd of HC13 toward p300 determined by MST. (I) IC50 of HC13 determined by FP competition assay using FITC-labeled HIF-1α CTAD as the fluorophore. (J) Western blot analysis of VEGF-A, c-Met, and HIF-1α proteins in HeLa cells under deferoxamine mesylate (DFO) (300 μM)-induced hypoxia treated with HC11, HC13, HC14, HCR, and ALA at 20 μM. (K) Pull-down experiment by using biotin-labeled HC11, HC13, and HC14 at 20 μM to bind p300.

Figure 5.
Figure 5.

(A) Venn diagram depicting unique and shared transcripts downregulated in hypoxic cells treated with the sulfonyl-γ-AApeptides HC11, HC13, and HC14. (B) The HIF-1 signaling pathway regulated by sulfonyl-γ-AApeptides. **Genes that were downregulated in each of the three AA peptide-treated samples are in bold. (C) Heatmap displaying the transcriptomic profiles of all major genes involved in the HIF-1 signaling pathway for each of the five samples is presented above. Green shading corresponds to downregulated genes, while red shading is indicative of upregulated genes. Normalized RPKM, which is calculated using the formula (raw RPKM value – mean RPKM)/SD (RPKM), was used to plot the heatmap. Upregulated and downregulated expression levels are colored red and green respectively. Most of the genes are upregulated by hypoxia but downregulated by any of three compounds.

Figure 6.
Figure 6.

Cell permeability of FITC-labeled HCR, HC11, HC13, and HC14 at 1, 5, 10, and 20 μM with incubation with HeLa cells for 24 h. Blue: nucleus. Green: FITC-labeled sulfonyl-γ-AApeptides. Scale bar: 20 μm.

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