Challenges in Targeting a Basic Helix–Loop–Helix Transcription Factor with Hydrocarbon-Stapled Peptides

Abstract

Basic helix–loop–helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein–protein and protein–DNA interactions mediated by α-helices. Thus, α-helical decoys have been proposed as potential targeted therapies for pathologic bHLH transcription. Here, we developed a library of stabilized α-helices of OLIG2 (SAH-OLIG2) to test the capacity of hydrocarbon-stapled peptides to disrupt OLIG2 homodimerization, which drives the development and chemoresistance of glioblastoma multiforme, one of the deadliest forms of human brain cancer. Although stapling successfully reinforced the α-helical structure of bHLH constructs of varying length, sequence-specific dissociation of OLIG2 dimers from DNA was not achieved. Re-evaluation of the binding determinants for OLIG2 self-association and stability revealed an unanticipated role of the C-terminal domain. These data highlight potential pitfalls in peptide-based targeting of bHLH transcription factors given the liabilities of their positively charged amino acid sequences and multifactorial binding determinants.

The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

“…As described previously, most DNA-binding domains of the CSRs contain a helixturn-helix (HTH) motif, a helix-loop-helix (HLH) motif, or a ribbon-helix-helix (RHH) motif (35)(36)(37). Generally, in CSRs, one of the ␣-helices is inserted into the major groove of the target DNA and thus binds to the DNA backbone (38).…”

Peptide therapeutics that directly target transcription factors

“…( ). To generate an OLIG2 inhibitor, a library of short peptides based on the sequences of either helix 1 or helix 2 of the OLIG2 HLH were synthesized . These synthetic peptides were stabilized using the hydrocarbon stapling method to enhance the helical structure of peptides by forming covalent bridges between amino acid residues .…”

“…The resulting series of peptides—stabilized α‐helices of OLIG2 (SAH‐OLIG2)—were hypothesized to bind to the OLIG2 HLH, thereby disrupting OLIG2 homodimerization and DNA binding. However, these peptides were not capable of specifically interfering with the OLIG2‐DNA interaction in EMSA, despite their having increased stability, as shown by circular dichroism spectroscopy . Interestingly, after an attempt to determine why SAH‐OLIG2 was not effective, the authors discovered that a region C‐terminal to the OLIG2 bHLH was playing an unexpected role in OLIG2 dimerization, suggesting that this C‐terminal may be an alternative target for peptide therapeutics.…”

“…This paper illustrates a major point in rational design of molecules; rational design is limited by our knowledge of the target, and that knowledge is often built through efforts like these studies. As mentioned earlier, other scientists, such as Wang and colleagues, have approached the field using nonrational methods such as phage display [reviewed in Ref.…”

Editorial on “Transcription factor SPZ1 promotes TWIST-mediated epithelial—mesenchymal transition and oncogenesis in human liver cancer”

“…Although the authors demonstrate that SPZ1 is a potential target for cancer therapy, targeting bHLH transcription factors with peptides is challenging (14). In addition, off target effects on the many other critical bHLH transcription factors are likely.…”