Synthetic peptide-based drugs enable efficient protein targeting and overcome some limitations of large antibody- and small molecule-drugs. To further improve the potential of peptide-based drugs, scientists at the Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, have implemented a new peptide design that leverages their learnings from polyglutamine (PolyQ) containing proteins, such as the androgen receptor (AR) and huntingtin. Their strategy relies on introducing peptide modifications that promote hydrogen bond formation at critical positions, which support stable alpha-helical structures (Escobedo et al. 2019 and 2022).
Targeting biological processes with peptide therapeutics overcomes several limitations associated with small molecule drugs. For instance, using peptide-based drugs diminishes toxicity risks commonly associated with small molecule-derived metabolites. In addition, peptide-based drugs are more efficacious, selective, and specific, providing better opportunities to target proteins relevant to various disease states, such as inflammation, neurodegeneration, cancer, and more. Moreover, unlike larger biological drugs, such as antibody drugs, which are more effective at reaching extracellular targets, peptides may be chemically modified or delivered with specific carriers enabling efficient intracellular protein targeting (La Manna et al. 2018).
Prior work by Escobedo et al. demonstrated that polyglutamine tracts, as commonly found in some transcription factors, impart a helical conformation that depends on the formation of unconventional hydrogen bonds. These interactions occur between donor groups from glutamine side chains and main chain carbonyl groups (Escobedo et al. 2019).
In their new work, Escobedo and colleagues have leveraged these unconventional hydrogen bond interactions to design stable helical peptides with high-affinity binding to globular proteins (Escobedo et al. 2022).
The team synthesized a series of peptides with conserved amino acid composition to learn how unconventional hydrogen bond interactions could be best leveraged in peptide design. Each peptide had a characteristic donor-acceptor (i.e., glutamine-leucine) pair arrangement and number, with some containing as many as three glutamine-leucine pairs. All peptides were confirmed to be monomeric, and analysis through circular dichroism spectroscopy revealed the optimal relative positioning of glutamine-leucine pairs supporting maximal interaction strength. Moreover, by analyzing the helicity of peptides having multiple glutamine-leucine pairs in series, the team found that helical propensity was increased with the number of donor-acceptor pairs, being maximal for those having a total of three concatenated pairs (i.e., six glutamine-leucine interactions in series) and referred to as a (P3-7)3 peptide.
Circular dichroism spectroscopy and NMR analysis demonstrated that the (P3-7))3 peptide’s helicity is thermostable. Moreover, of significance to the potential therapeutic implementation of this peptide design strategy, the (P3-7)3 peptide conserved its helical properties under physiologically relevant conditions, was resistant to proteolytic cleavage and could be internalized by cells in vitro.
Ultimately, Escobedo and colleagues wanted to test the efficiency of such helical peptide scaffolds in targeting specific globular proteins. To this end, the team replaced specific residues within the (P3-7)3 peptide to target the carboxy-terminal domain of the RNA polymerase II associating protein 74 (RAP74). Amino acids for the new “δ” peptide sequence were selected based on the TFIIF-associating CTD phosphatase (FCP1) binding motif, a RAP74 interacting protein. Additionally, a RAP74 targeting peptide was designed having decreased helicity, “δctrl” as a control. Analysis of the interaction between RAP74 and the two modified peptides confirmed high-affinity binding, at the micromolar range, between the δ peptide and RAP74 and no interaction with the control. Moreover, similarly, higher affinity binding was observed between a (P3-7)3 peptide modified to emulate the androgen receptor’s RAP74 interacting domain, as opposed to the control peptide. Therefore, through these studies, the team demonstrated the relevance of helicity for effective interaction between peptides and globular protein domains.
Overall, by applying peptide design rules learned from natural polyQ-bearing proteins, Escobedo and colleagues have created a platform that can be customized for the effective and potential therapeutic targeting of globular proteins.
Escobedo, A., et al. (2019). Side chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor. Nat Commun https://doi.org/10.1038/s41467-019-09923-2
Escobedo, A., et al. (2022). A glutamine-based single α-helix scaffold to target globular proteins. Nat Commun https://doi.org/10.1038/s41467-022-34793-6
La Manna, S., di Natale, C., Florio, D., & Marasco, D. (2018). Peptides as Therapeutic Agents for Inflammatory-Related Diseases. International Journal of Molecular Sciences https://doi.org/10.3390/IJMS19092714