Discovery of Potent Degraders of the Dengue Virus Envelope Protein

Abstract

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here we report the development of small molecule degraders of the envelope (E) protein of dengue virus. We developed two classes of bivalent E-degraders, linking two previously reported E-binding small molecules, GNF-2 and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4^CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E degrader with ABL inhibition while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof-of-concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class antiviral drugs.

Keywords: antivirals; dengue; envelope protein; infection; protein degradation.

Figure 1.

Design of PROTACs targeting the dengue E protein. A) Chemical structures of GNF-2 and CVM-2-12-2. B) Optimal docking poses of dengue E protein (PDB: 1OKE) with GNF-2. C) Optimal docking poses of dengue E protein (PDB: 1OKE) with CVM-2-12-2. The E protein is shown as a ribbon cartoon and the key residues forming hydrogen bonds are represented as sticks. Docking studies were performed with Schrödinger, and models were prepared with PyMOL.

Figure 2.

Screening of GNF-2 based degraders. A) Schematic representation of the viral infection assay utilized to evaluate E protein degradation and antiviral activity. Created on BioRender.com with permission. B) and C) Immunoblot analyses of dengue E abundance following treatment of DENV2-infected Huh7.5 cells with GNF-2-based degraders at 5 and 10 μM with and without pretreatment of 5 μM lenalidomide.

Figure 3.

Screening of CVM-2-12-2 based degraders. A) Schematic representation of the viral infection assay utilized to evaluate E protein degradation and antiviral activity. B) and C) Immunoblot analyses of dengue E abundance following treatment of DENV2-infected Huh7.5 cells with CVM-2-12-2-based degraders at 5 and 10 μM with and without pretreatment of 5 μM lenalidomide.

Figure 4.

A) Cellular CRBN engagement assay of ZXH-2-107 and ZXH-8-004 at 10 μM. B) E protein cellular thermal shift assay (CETSA) for E inhibitors and degraders. The quantitative analysis of immunoblot of sample from 59.7 °C is performed with normalization of GAPDH.

Figure 5.

CRBN-dependence of ZXH-2-107- and ZXH-8-004-induced degradation of dengue E. A) Immunoblot analysis of E in DENV2-infected Huh 7.5 cells treated with ZXH-2-107 or ZXH-2-107-Neg for 24 h. B) Immunoblot analysis of E in DENV2-infected Huh 7.5 cells treated with ZXH-8-004 or ZXH-8-004-Neg for 24 h. C) Immunoblot analysis of E in DENV2-infected Huh 7.5 cells treated with GNF-2 or CVM-2-12-2 for 24 h. Semi-quantitative analysis was performed using the GAPDH signal for normalization.

Figure 6.

A) Proteome-wide degradation selectivity of ZXH-8-004 at a dose of 3 μM in MOLT4 cells after 5 hour treatment. B) Cellular GSPT1-induced degradation of ZXH-8-004 and CC-90009 assessed by reporter assay.

Figure 7.

Antiviral activities of E degraders, negative controls, and parental inhibitors against DENV1, 2, 3, and 4 in Huh 7.5 cells at 2.5 μM and 5 μM. The yield of progeny virus in culture supernatants at 24 hours post-infection was quantified by viral plaque formation assay. Graphs show viral titer as % of the DMSO-treated, DENV2-infected controls.