Treatment of leukemia cells with these hydrocarbon-stapled peptides resulted in genome-wide suppression of Notch-activated genes. Use of stapled peptides to target the interaction of transcription factors was reported in earlier research of Moellering et al., who designed hydrocarbon-stapled alpha-helical peptides targeting the critical protein–protein interface in the Notch transcription complex. Transcription factors, which play a major role in regulation of cell state and neoplastic growth, are considered prime candidates as drug targets. However, these alternatives to hydrocarbon stapling involve cross-links that are both polar and pharmacologically labile. Stabilization of alpha-helices may be achieved by lactam bridges, disulfide bridges, H-bond surrogates, and alpha/beta peptides.

These researchers suggested that hydrocarbon stapling of native peptides could prove useful for modulation of protein–protein interactions (PPIs) in many signaling pathways. One of these stapled peptides specifically activated the apoptotic pathway to kill leukemia cells.

The group of Verdine first used the term “stapled peptides” to describe peptides mimicking the BH3 helical region, which had increased affinity to multidomain BCL-2 member pockets and improved pharmacological properties. Control of apoptosis in BCL-2 proteins is mediated through an alpha-helical segment, BH3. This finding was applied to create peptides which interfered with regulation of apoptosis in tumor cells by BCL-2 family proteins and their interaction. first demonstrated that an all-hydrocarbon cross-linking system could greatly increase the helical propensity and metabolic stability of peptides. The general concept of peptide stapling was introduced about two decades ago by Verdine et al. Commonly used linkers are hydrocarbon chains introduced as side chains of unnatural amino acid residues, which are then chemically bonded together during the final stages of peptide synthesis, or side chains of Asp and Glu residues joined to side chains of Arg and Lys residues via lactamization of the side chains. Stapling of alpha helices usually involves linking one amino acid residue with the equivalently located residue one turn (residue i linked to residue i+4) or two turns (residue i linked to residue i+7) further along the helix. Increased helicity renders them more resistant to proteolytic breakdown by host proteases furthermore, stapling increases potency and often improves cell penetration. Stapling can be applied to enhance the alpha-helicity of peptides, which would otherwise adopt a random coil conformation in solution. Stapling involves such chemical modifications designed to constrain the peptide chain in the secondary structure of the native conformation, and the peptide products are known as stapled peptides.

Because peptides can lose their shape when taken out of context, developing chemical interventions to stabilize their bioactive structure remains an active area of research. Loss of secondary structure usually means loss of biological function. Short peptides, when isolated from the parent protein, lose their secondary structure and exist in solution as unstructured, linear molecules. These stapled peptides, which mimic Helix 1 of the human ACE2 receptor, have demonstrated mixed ability to prevent infection with SARS-CoV-2 in cell-based studies.

Several independent research initiatives have investigated the inhibitory effect of stapled peptides on binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, to its receptor, angiotensin-converting-enzyme 2 (ACE2). The potential use of stapled peptides as inhibitors of viral entry, and therefore as antiviral therapeutics, has been established for several important viruses causing disease in humans, such as the human immunodeficiency virus type 1 (HIV-1), respiratory syncytial virus (RSV), and Middle East Respiratory Syndrome (MERS) coronavirus. Stapled peptides have been investigated as potential modulators of protein–protein interactions for over two decades. Stapled peptides are synthetic peptidomimetics of bioactive sites in folded proteins which carry chemical links, introduced during peptide synthesis, designed to retain the secondary structure in the native protein molecule.

Stapled binding