title: Development of an Antiviral PROTAC Using the Example of the Transcription Factor VP30 of Ebola Virus and the Main Protease MPro of SARS Coronavirus Type 2
title: Entwicklung eines antiviralen PROTAC am Beispiel des Transkriptionsfaktors VP30 des Ebola virus und der Hauptprotease MPro des SARS Coronavirus Typ 2
titleType: AlternativeTitle
Creators: name: Veeck, Christopher
Publisher: name: Philipps-Universität Marburg
Year: 2025
Subjects: SARS-CoV-2
Drug Design
Medical sciences Medicine
Medizin
PROTAC
Ebola virus
Targeted Protein Degradation

description: Targeted protein degradation (TPD) utilizes heterobifunctional molecules called proteolysis-targeting chimera (PROTAC) to force an interaction between cellular E3 ligases and a target protein (usually an enzyme), leading to polyubiquitination and thus, degradation of the target protein in the proteasome. In order to expand the antiviral arsenal, this work aims to investigate the establishment of targeted protein degradation as a new antiviral therapy. All known antiviral PROTACs used ligands (warhead) that target the catalytic pocket of the target protein. In the present work, it was investigated, whether the development of PROTACs is also possible with a warhead that binds allosterically to the target protein in order to also enable viral structural proteins as targets. The aim of the present work was the development of an antiviral PROTAC with 1) the characterization of the target protein, 2) the identification of ligands that can be used as warhead, and finally 3) the development and optimization of the first antiviral PROTAC that uses an allosteric warhead to span the complete cycle of PROTAC development. 

Several proteins of the Ebola virus and SARS-CoV-2 have been investigated as potential targets for targeted protein degradation. Orthoebolaviruses such as Ebola virus (EBOV) can cause severe hemorrhagic fevers with high mortality rates, and to date there are no options for antiviral therapies other than specific antibodies. The phosphoprotein VP30 is a transcription factor that is essential for the transcription of the viral genome and thus for the replication of the virus. This property makes it a promising target for antiviral therapy. Since VP30 is active as a homo-oligomer, the self-interaction of the protein was characterized. Based on the crystal structure of the VP30 C-terminus, bioinformatic programs as well as chemical cross-linking, SEC-MALS, native mass spectrometry and functional analyses were used to show that in addition to the hexamerization domain of VP30, a second, C-terminal dimerization domain of the protein is essential its transcription-activating function. Therefore, VP30 dimerization represents a promising target for the development of interaction inhibitors as a new antiviral strategy. Furthermore, the main protease MPro of SARS-CoV-2, causative agent of the 2020 - 2023 pandemic, has been identified as a possible target protein. Betacoronaviruses such as SARS-CoV-2 are respiratory viruses that can cause severe airway inflammation and lung failure. The MPro plays a crucial role for the virus in the early phase of the replication cycle, which is why it is a popular target for inhibitors. 

To identify a ligand that can be used as a warhead for the development of an antiviral PROTAC, a fragment library of 94 compounds was used in a thermal-shift assay-based prescreening. Among the fragments tested, two compounds proved to be promising VP30-binding ligands that were inhibitory active in surrogate systems for EBOV RNA synthesis. In particular, fragment J23 showed high antiviral activity with low cytotoxicity (IC50: 8.67 µM) after 48 hours. Both J23 and J84 can therefore also be used as warheads for the development of anti-VP30-PROTACs or small molecule inhibitors. 

In order to investigate the suitability of various viral proteins, including EBOV VP30 and the SARS-CoV-2 MPro, for targeted protein degradation, the viral proteins were fused to FKBP12, which served as a degradation signal, and examined for degradability using anti-FKBP12 PROTACs. This resulted in a strong degradability of FKBP12-MPro, whereas FKBP12-VP30, as well as other Ebola virus structural 
proteins, could not be degraded. As a result, the development of anti-VP30 PROTACs was abandoned. As a result, pelitinib, which is known to bind to MPro, was used as a warhead against MPro. In addition, medicinal chemically synthesized derivatives of pelitinib were produced, so that ultimately ten different PROTACs with different linker lengths and configurations were investigated. Of these, LLP019 
proved to be a potent degrader of ectopically expressed MPro in HEK293 cells. Inhibition of the cellular ubiquitination machinery prevented LLP019-induced degradation of MPro, confirming the proposed mode of action of LLP019 via the proteasome. Treatment of SARS-CoV-2 infected Calu3 cells with LLP019 showed a reduction in viral titers by about one log level (IC50: 17.67 µM) 48 hours after 
infection. The resulting bell-shaped dose-dependent inhibition, also known as the hook effect, is characteristic of PROTACs. This work demonstrates the successful development of an antiviral PROTAC that utilizes an allosteric warhead and therefore does not target the catalytic pocket of the target protein, highlighting the feasibility of GPD in an antiviral context. 

In summary, this work makes two important contributions to the development of antiviral inhibitors. The dimerization of EBOV VP30 was identified and characterized. The formation of VP30 dimers is essential for the formation of higher oligomers and thus for the function of VP30. VP30-binding molecules were able to inhibit the function of VP30. This work represents the first successful discovery 
of an antiviral PROTAC utilizing an allosteric warhead, laying the foundation for the further development of targeted protein degradation as an antiviral strategy.