Periodic Reporting for period 1 - MEDICATE (Molecular dissection of viral genomes for future antiviral treatments)
Reporting period: 2023-01-01 to 2025-06-30
Viruses are obligatory pathogens that significantly impact global health. Most current antiviral drugs target viral enzymes. We propose that previously unrecognized viral transmembrane proteins could serve as valuable pharmaceutical targets.
By dissecting viral genomes, we aim to identify potential transmembrane segments through a defined workflow. We will predict their function in silico (WP1) as either (A) internalizing transmembrane proteins or (B) ion channels. Promising segments will be expressed, functionally characterized, and evaluated in proof-of-modality assays (WP2). The internalizing proteins will be tested for their ability to transfer a molecular Trojan horse, a toxin payload, into cells using a universal fusion-toxin protein (Modality A). Potential viral ion channels (viroporins) will be assessed for their ability to mediate a current (Modality B).
In WP3, fusion toxin proteins will be produced for internalizing targets that pass WP1-2 attrition. These toxins, co-internalized with viral targets, will prevent long-term pathologies by eradicating the virus. Viroporins will be screened for inhibition using ion channel drugs already approved for other purposes, ensuring fast market access through drug repurposing and expediting the pipeline for acute viral illness prevention and treatment.
Finally, in WP4, the identified viral target/drug pairs will be tested for antiviral efficacy using virus-infected human organoids and/or discarded human organs.
This innovative project, while high-risk, holds the potential for significant gains given the unmet medical need for effective antiviral therapeutics. The greatest prospect lies in the potential for groundbreaking discoveries regarding virally encoded transmembrane proteins, bridging basic virology, molecular pharmacology, structural biology, and early drug discovery.
By dissecting viral genomes, we aim to identify potential transmembrane segments through a defined workflow. We will predict their function in silico (WP1) as either (A) internalizing transmembrane proteins or (B) ion channels. Promising segments will be expressed, functionally characterized, and evaluated in proof-of-modality assays (WP2). The internalizing proteins will be tested for their ability to transfer a molecular Trojan horse, a toxin payload, into cells using a universal fusion-toxin protein (Modality A). Potential viral ion channels (viroporins) will be assessed for their ability to mediate a current (Modality B).
In WP3, fusion toxin proteins will be produced for internalizing targets that pass WP1-2 attrition. These toxins, co-internalized with viral targets, will prevent long-term pathologies by eradicating the virus. Viroporins will be screened for inhibition using ion channel drugs already approved for other purposes, ensuring fast market access through drug repurposing and expediting the pipeline for acute viral illness prevention and treatment.
Finally, in WP4, the identified viral target/drug pairs will be tested for antiviral efficacy using virus-infected human organoids and/or discarded human organs.
This innovative project, while high-risk, holds the potential for significant gains given the unmet medical need for effective antiviral therapeutics. The greatest prospect lies in the potential for groundbreaking discoveries regarding virally encoded transmembrane proteins, bridging basic virology, molecular pharmacology, structural biology, and early drug discovery.
Modality (Track) A: Internalizing Transmembrane Proteins
In the first period, progress was made in studying virus-encoded internalizing transmembrane proteins, specifically viral G protein-coupled receptors (vGPCRs) from herpesviruses. These vGPCRs can bind to host chemokines and internalize either in a ligand-dependent or constitutive manner, aiding in immune evasion and presenting opportunities for drug targeting. The focus has been on two vGPCRs, which have been SNAP-tagged for real-time internalization studies and will be analyzed using nanoLuc bystander BRET-based assays.
Modality (Track) B: Viroporins
Viroporins, viral transmembrane proteins forming ion channels, are promising antiviral drug targets. The MEDICATE project developed a novel assay system, ViPoFluX, for rapid and large-scale screening of viroporins. Initial screenings identified three viroporins with strong pore activity. These were tested with generic inhibitors and a small inhibitor library. Ongoing efforts include screening with a larger ion channel inhibitor library and developing a pipeline in collaboration with DTU for further testing.
Additionally, five new viroporin genes are being probed for basic activity. A state-of-the-art overview of viroporins was published in collaboration with IUPHAR, and an in-depth analysis of the SH protein from the mumps virus was accepted for publication.
In the first period, progress was made in studying virus-encoded internalizing transmembrane proteins, specifically viral G protein-coupled receptors (vGPCRs) from herpesviruses. These vGPCRs can bind to host chemokines and internalize either in a ligand-dependent or constitutive manner, aiding in immune evasion and presenting opportunities for drug targeting. The focus has been on two vGPCRs, which have been SNAP-tagged for real-time internalization studies and will be analyzed using nanoLuc bystander BRET-based assays.
Modality (Track) B: Viroporins
Viroporins, viral transmembrane proteins forming ion channels, are promising antiviral drug targets. The MEDICATE project developed a novel assay system, ViPoFluX, for rapid and large-scale screening of viroporins. Initial screenings identified three viroporins with strong pore activity. These were tested with generic inhibitors and a small inhibitor library. Ongoing efforts include screening with a larger ion channel inhibitor library and developing a pipeline in collaboration with DTU for further testing.
Additionally, five new viroporin genes are being probed for basic activity. A state-of-the-art overview of viroporins was published in collaboration with IUPHAR, and an in-depth analysis of the SH protein from the mumps virus was accepted for publication.
In modality B, the ViPoFluX assay system was developed to overcome the limitations of previous viroporin activity tests. This system is now being optimized.
Further refinements are needed before the inclusion of new potential viroporins and before library screening.
Further refinements are needed before the inclusion of new potential viroporins and before library screening.