Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent infection in humans, but under conditions of immune suppression, it may reactivate and contribute to severe diseases, including Kaposi's sarcoma (KS) and B-cell malignancies. The KSHV genome encodes a single G protein-coupled receptor (GPCR), open reading frame 74 (ORF74), which shows homology to human chemokine receptors. Since its identification in 1996, ORF74 has subsequently been shown to interact with a broad range of human CXC chemokines, as well as CCL1 and the viral chemokine vCCL2. Compared with many human chemokine receptors, ORF74 displays high basal activity. These properties allow ORF74 to deregulate host cellular pathways through constitutive and chemokine-modulated signaling. In this study, we evaluated several human chemokines that, to our knowledge, had not previously been tested in ORF74-dependent cellular assays. Whereas CXCL9, CXCL14, CXCL16 and CXCL17 did not interact with ORF74, CXCL13 was identified as an additional ORF74 agonist and CXCL11 as an inverse agonist. CXCL13 dose-dependently induced ORF74-mediated Ca2+ release, β-arrestin1/2 recruitment and chemotaxis, and enhanced basal nuclear factor κB (NF-κB) activity in ORF74-expressing cells. In contrast, CXCL11 showed no detectable ORF74 agonist activity in the calcium mobilization or chemotaxis assay, but antagonized CXCL1-induced responses in both readouts. CXCL11 also elicited inverse agonist-like responses in β-arrestin1/2 recruitment assays and reduced basal NF-κB signaling. Our study thus reveals CXCL13 and CXCL11 as two additional chemokine ligands for ORF74, further expanding the pharmacological profile of this viral GPCR.
Qianqian Kong, Brent Van Bosstraeten, S. de Jonghe et al.· Biochemical Pharmacology· 0 citations
Lens epithelium‐derived growth factor p75 (LEDGF/p75) is a chromatin reader that recognizes di‐ or trimethylated Lys36 of histone H3 (H3K36me2/3)‐modified nucleosomes and is implicated in diverse diseases, including cancer and human immunodeficiency virus (HIV) infection. Inhibiting the interaction between the Pro‐Trp‐Trp‐Pro (PWWP) domain of LEDGF and chromatin through a small‐molecule drug presents an attractive therapeutic opportunity, but the compounds developed to date bind entirely within the small H3K36me2/3 pocket and achieve only modest affinity. Here, we report de novo computational design and structural validation of proteinaceous binders that engage both this canonical pocket and adjacent DNA‐interacting surfaces of the PWWP domain. Using a hotspot‐driven workflow integrating RFdiffusion, ProteinMPNN, AlphaFold, molecular dynamics simulations and manual structural assessment, we generated four protein designs that were subjected to experimental validation. Biophysical analysis confirmed that one designed binder had a low‐micromolar affinity for the LEDGF PWWP domain. Another designed binder revealed unexpected homodimerization which apparently interfered with its binding to the target in solution. Nevertheless, this binder could be co‐crystallized with the PWWP domain. The resulting atomic structure at 2.1 Å resolution confirms correct engagement of the intended binding interface. This crystal structure enabled the construction of an expanded pharmacophore model that can instruct the design of next‐generation small‐molecule or peptide‐based inhibitors targeting the LEDGF PWWP domain and related epigenetic readers. These results demonstrate that modern in silico design pipelines can directly yield functional proteinaceous binders without the need for additional experimental screening using phage display or related technologies.
Thibault Vantieghem, Julie Delepine, Sam Noppen et al.· Protein Science· 0 citations
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