Discovery of Pentacyclic Triterpenoid PROTACs as a Class of Effective Hemagglutinin Protein Degraders

Abstract

Influenza hemagglutinin that drives viral entry into cells via the membrane fusion process is an up-and-coming antiviral drug target. Herein, we described for the first time the design, synthesis, and biological characteristics of a new class of pentacyclic triterpenoid-based proteolysis targeting chimeras (PROTACs) to enhance the degradation of hemagglutinin target. Among these PROTACs, V3 showed the best degradation effect on the hemagglutinin with a median degradation concentration of 1.44 μM in a ubiquitin and proteasome-dependent manner and broadspectrum anti-influenza A virus activity but not affected the entry of influenza virus. Moreover, intravenous injection of V3 protected mice against influenza A virus-induced toxic effects. Further diazirine-containing photo-crosslinking mass spectrometric analysis of hemagglutinin complexes indicated crosslinking to Asn15, Thr31, and Asn27, a novel target of hemagglutinin. Taken together, our data revealed that oleanolic acid-based PROTACs could degrade hemagglutinin protein, providing a new direction toward the discovery of potential anti-influenza drugs.

Natural Product-Inspired Targeted Protein Degraders: Advances and Perspectives

“…However, anti-influenza virus entry potency was not demonstrated at the tested concentration of 25 μM, indicating that there was a new HA binding site. This suggestion was further confirmed by diazirine-containing photo-cross-linking mass spectrometry analysis . Importantly, 73 not only decreased the virus titer in infected cells but also protected mice from death and weight loss caused by influenza virus infection while exhibiting moderate oral bioavailability .…”

“…The Zhou group designed two series of OA PROTACs containing OA and CRBN or VHL recruiters linked by oligo­(ethylene glycol) (OEG) linkers with different lengths. These compounds exhibited different HA degradation abilities in 293T cells infected by influenza virus . The most effective VHL–OA PROTAC, V3 ( 73 ) (Figure ), exhibited a potent HA-degrading effect (DC 50 = 1.44 μM) and broad-spectrum anti-influenza A virus activity (EC 50 = 4.53–8.98 μM).…”

“…The new compound exhibited more selective and sustained degradation of CRABP-II compared SNIPER-2 (CRABP-II was effectively degraded by SNIPER-4 at 3 μM in HT1080 cells for 6 h) without the degradation of cIAP1. 60 Another molecule, SNIPER-11 (19), was prepared by replacing bestatin with the more potent cIAP1 recruiter MV1 (1 and 3 μM in HT1080 cells, 6 h). 61,62 This molecule showed more potent cytotoxic and caspase-activating effects than SNIPER-2 in neuroblastoma IMR-32 cells and efficiently degraded both cIAP1 and CRABP-II through a proteasomal pathway.…”

“…This suggestion was further confirmed by diazirine-containing photo-cross-linking mass spectrometry analysis . Importantly, 73 not only decreased the virus titer in infected cells but also protected mice from death and weight loss caused by influenza virus infection while exhibiting moderate oral bioavailability . Overall, this is the first PROTAC used as an anti-influenza virus drug, expanding the application of OA in PROTAC research and providing a new strategy for using new therapeutic agents to treat influenza infection.…”

“…The first proof-of-concept study with PROTACs was performed with a natural polyketide ovalicin-derived molecule in 2001 . Natural products (NPs) are widely utilized in the field of TPD, especially as PROTACs and molecular glues, to provide ligands that recruit new E3 ligases (such as nimbolide, which binds to RNF114), new POI-targetting warheads (such as oleanolic acid (OA), which target hemagglutinin), and new molecular glues (such as asukamycin). The use of NP-inspired targeted protein degraders has been confirmed as a promising strategy to treat cancer, inflammation, nonalcoholic fatty liver disease, etc . ,, Nevertheless, although the development of NP-inspired targeted protein degraders is currently still in its infancy, it has indicated great prospects for the application of NP resources in TPD.…”