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

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.

Modulating Angiogenesis by Proteomimetics of Vascular Endothelial Growth Factor.

Angiogenesis, formation of new blood vessels from the existing vascular network, is a hallmark of cancer cells that leads to tumor vascular proliferation and metastasis. This process is mediated through the binding interaction of VEGF-A with VEGF receptors. However, the balance between pro-angiogenic and anti-angiogenic effect after ligand binding yet remains elusive and is therefore challenging to manipulate. To interrogate this interaction, herein we designed a few sulfono-γ-AA peptide based helical peptidomimetics that could effectively mimic a key binding interface on VEGF (helix-α1) for VEGFR recognition. Intriguingly, although both sulfono-γ-AA peptide sequences V2 and V3 bound to VEGF receptors tightly, in vitro angiogenesis assays demonstrated that V3 potently inhibited angiogenesis, whereas V2 activated angiogenesis effectively instead. Our findings suggested that this distinct modulation of angiogenesis might be due to the result of selective binding of V2 to VEGFR-1 and V3 to VEGFR-2, respectively. These molecules thus provide us a key to switch the angiogenic signaling, a biological process that balances the effects of pro-angiogenic and anti-angiogenic factors, where imbalances lead to several diseases including cancer. In addition, both V2 and V3 exhibited remarkable stability toward proteolytic hydrolysis, suggesting that V2 and V3 are promising therapeutic agents for the intervention of disease conditions arising due to angiogenic imbalances and could also be used as novel molecular switching probes to interrogate the mechanism of VEGFR signaling. The findings also further demonstrated the potential of sulfono-γ-AA peptides to mimic the α-helical domain for protein recognition and modulation of protein-protein interactions.

Rational Design and Synthesis of Right-Handed d-Sulfono-γ-AApeptide Helical Foldamers as Potent Inhibitors of Protein-Protein Interactions.

Novel unprecedented helical foldamers have been effectively designed and synthesized. The homogeneous right-handed d-sulfono-γ-AApeptides represent a new generation of unnatural helical peptidomimetics, which have similar folding conformation to α-peptides, making them an ideal molecular scaffold to design α-helical mimetics. As demonstrated with p53-MDM2 PPI as a model application, the right-handed d-sulfono-γ-AApeptides reveal much-enhanced binding affinity compared to the p53 peptide. The design of d-sulfono-γ-AApeptides may provide a new and alternative strategy to modulate protein-protein interactions.

α-Helix-Mimicking Sulfono-γ-AApeptide Inhibitors for p53-MDM2/MDMX Protein-Protein Interactions.

The use of peptidomimetic scaffolds is a promising strategy for the inhibition of protein-protein interactions (PPIs). Herein, we demonstrate that sulfono-γ-AApeptides can be rationally designed to mimic the p53 α-helix and inhibit p53-MDM2 PPIs. The best inhibitor, with Kd and IC50 values of 26 nM and 0.891 µM toward MDM2, respectively, is among the most potent unnatural peptidomimetic inhibitors disrupting the p53-MDM2/MDMX interaction. Using fluorescence polarization assays, circular dichroism, nuclear magnetic resonance spectroscopy, and computational simulations, we demonstrate that sulfono-γ-AApeptides adopt helical structures resembling p53 and competitively inhibit the p53-MDM2 interaction by binding to the hydrophobic cleft of MDM2. Intriguingly, the stapled sulfono-γ-AApeptides showed promising cellular activity by enhancing p53 transcriptional activity and inducing expression of MDM2 and p21. Moreover, sulfono-γ-AApeptides exhibited remarkable resistance to proteolysis, augmenting their biological potential. Our results suggest that sulfono-γ-AApeptides are a new class of unnatural helical foldamers that disrupt PPIs.

Inhibition of ß-catenin/B cell lymphoma 9 protein-protein interaction using α-helix-mimicking sulfono-γ-AApeptide inhibitors.

The rational design of α-helix-mimicking peptidomimetics provides a streamlined approach to discover potent inhibitors for protein-protein interactions (PPIs). However, designing cell-penetrating long peptidomimetic scaffolds equipped with various functional groups necessary for interacting with large protein-binding interfaces remains challenging. This is particularly true for targeting ß-catenin/BCL9 PPIs. Here we designed a series of unprecedented helical sulfono-γ-AApeptides that mimic the binding mode of the α-helical HD2 domain of B Cell Lymphoma 9 (BCL9). Our studies show that sulfono-γ-AApeptides can structurally and functionally mimic the α-helical domain of BCL9 and selectively disrupt ß-catenin/BCL9 PPIs with even higher potency. More intriguingly, these sulfono-γ-AApeptides can enter cancer cells, bind with ß-catenin and disrupt ß-catenin/BCL9 PPIs, and exhibit excellent cellular activity, which is much more potent than the BCL9 peptide. Furthermore, our enzymatic stability studies demonstrate the remarkable stability of the helical sulfono-γ-AApeptides, with no degradation in the presence of pronase for 24 h, augmenting their biological potential. This work represents not only an example of helical sulfono-γ-AApeptides that mimic α-helix and disrupt protein-protein interactions, but also an excellent example of potent, selective, and cell-permeable unnatural foldameric peptidomimetics that disrupt the ß-catenin/BCL9 PPI. The design of helical sulfono-γ-AApeptides may lead to a new strategy to modulate a myriad of protein-protein interactions.