Synthesis and biological characterisation of cyclic peptide PROTACs of hypoxia inducible factor 1 alpha

Abstract

PROteolytic TArgeting Chimeras (PROTACs) are heterobifunctional chemical modalities that exploit the ubiquitin proteasome system (UPS) to degrade protein targets, including those considered ‘undruggable’. Despite the success of small molecule PROTACs, the application of cyclic peptides as targeting elements remains underexplored, despite their proven efficacy as protein-protein interaction (PPI) inhibitors.

This work reports the development of the first cyclic peptide-based PROTACs capable of inducing degradation of the Hypoxia Inducible Factor – 1 alpha (HIF-1 alpha) subunit, a transcription factor that functions through PPIs and is herald as a challenging drug target in oncology. Starting from the cyclic peptide cyclo-CLLFVY, a previously reported HIF-1 alpha inhibitor, a library of PROTACs was designed, incorporating short linkers to recruit either the Von Hippel Lindau (VHL) or Cereblon (CRBN) E3 ligase. Biophysical and cellular assays informed the design of second-generation PROTACs using the optimised cyclic peptide cyclo-CLLF(CF3)IF(Br) and longer unnatural amino acid linkers. The resulting cyclic peptide PROTACs successfully degraded HIF-1 alpha under both artificially induced hypoxia and physiologically relevant conditions, demonstrating their effectiveness against a key ‘undruggable’ target. This study is the first to convert a cyclic peptide derived from SICLOPPS screening into a functioning PROTAC and highlights the potential of incorporating unnatural amino acid linkers into peptide-based PROTACs.

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Identifiers

ORCID for Ali Tavassoli: orcid.org/0000-0002-7420-5063

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