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. Author manuscript; available in PMC: 2025 Apr 18.
Published in final edited form as: Cell Chem Biol. 2024 Mar 19;31(4):658–668.e14. doi: 10.1016/j.chembiol.2024.02.004

Figure 2. NanoBRET assay for PROTAC-mediated ubiquitination of HIV-1 Nef.

Figure 2.

A) Assay principle. Nef is fused to nano-Luciferase (Nef-nLuc) and co-expressed with a ubiquitin-Halo tag fusion protein (Ub-Halo) in 293T cells. PROTACs promote ligation of Ub-Halo to Nef-nLuc, which is detected by bioluminescence resonance energy transfer (BRET) to the Halo Tag. This model was produced using BioRender and the crystal coordinates for Nef (PDB: 6B72), NanoLuc (PDB: 5IBO) and HaloTag (PDB: 5Y2X). B) Assessment of candidate Nef PROTACs in the NanoBRET assay. Each compound was assayed in quadruplicate and the average 618 nm to 460 nm fluorescence ratios (BRET signal for Ub incorporation normalized to Nef-nLuc levels) were normalized to the DMSO control and are presented as z-scores ± SD (error bars smaller than data points). PROTACs with z-scores ≥ 1.5 (numbered red points) along with analog FC-13887 were advanced to orthogonal assays for Nef degradation and inhibition of Nef function. z-score = (x - μ)/σ, where x = each individual value, μ = mean value, and σ = the standard deviation.