Modulating ORF74 – Targeting the Viral GPCR of Human Herpesvirus 8

Objectives: Human herpesvirus 8 (HHV-8), also referred to as Kaposi's sarcoma-associated herpesvirus (KSHV), is the virus that leads to Kaposi's sarcoma, a type of cancer that commonly occurs in immunocompromised patients, often in combination with HIV infection.(1) The HHV-8 viral GPCR (vGPCR), ORF74, is linked to proliferative effects that cause Kaposi sarcoma cancer lesions(2). The development of an inverse agonist of ORF74 might be beneficial in the fight against Kaposi sarcoma. We therefore designed a virtual screening campaign to develop novel ORF74 modulators. Methods: Our workflow starts with the development of an ORF74 homology model using MOE(3), MODELLER(4), Rosetta(5), SwissModel(6), and Alphafold2(7). The plausibility of the models was evaluated based on their geometric properties and their stability in molecular dynamics simulations. After an optimal model was identified, protein-protein docking in Rosetta(5) was employed to identify the binding modes of chemokines CXCL8 (neutral binder)(8) and CXCL12 (negative modulator)(8). This was followed by the construction of structure-based 3D pharmacophores in LigandScout(9), with emphasis on interactions formed by the negative modulator CXCL12. These models were used to perform a 3D pharmacophore based virtual screening of 2.8 million commercially available small molecules. Virtual hits were further geometrically assessed by molecular docking using the software GOLD(10). Finally, the poses were visually inspected to identify potential hits based on their exhibited interactions as well as their positioning within the receptor. Results: Using a virtual screening campaign, we identified 17 potential modulators of ORF74. These will now be evaluated for activity in a competition binding assay and a calcium mobilization assay. Conclusion: ORF74, a G protein-coupled receptor highly relevant for the development of Kaposi's sarcoma as a druggable target that can be addressed using small organic molecules. A virtual screening campaign revealed interesting scaffolds that bear the potential to effectively modulate ORF74 activity and thus provide the basis for a therapy against HHV-8.