PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication

Abstract

The HIV-1 Nef accessory factor enhances the viral life cycle in vivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target. However, Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on an existing hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the cereblon E3 ubiquitin ligase thalidomide-binding domain in vitro and triggered Nef degradation in a T cell expression system. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo.

Keywords: CD4; HIV Nef; HIV-1; MHC-I; PROTACs; antiretroviral drugs; targeted degradation.

Figure 1.. Targeted degradation of HIV-1 Nef by a CRBN-based PROTAC.

The Cereblon (CRBN) ubiquitin E3 ligase complex (left) is a large multiprotein structure composed of RING-box protein 1 (RBX1), Cullin4 (CUL4), DNA damage binding protein 1 (DDB1), CRBN and an E2 subunit conjugated to ubiquitin (Ub). Heterobifunctional Nef PROTACs promote formation of a ternary complex between the HIV-1 Nef protein using existing hydroxypyrazole Nef-binding compounds (red) and the CRBN E3 complex via a CRBN ligand (exemplified by thalidomide, green). Ternary complex formation induces polyubiquitination of Nef and subsequent proteasomal degradation. The Nef PROTAC shown is analog FC-13182; favored positions for linker attachment on the Nef-binding moiety are indicated by the short black arrows. This model was produced using BioRender and structural coordinates for ubiquitin (PDB 1D3Z, NMR), HIV-1 Nef (PDB: 6B72, crystal) and an E3 ligase complex (PDB: 2HYE, crystal).

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

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.

Figure 3.. Nef PROTAC treatment restores cell surface CD4 and MHC-I expression in T cells.

The human T cell line CEM-T4 was engineered to express a Nef-eGFP fusion protein under the control of a doxycycline (Dox) inducible promoter. In the absence of Dox, these cells express endogenous CD4 and MHC-I on their surface; addition of Dox induces Nef-eGFP expression which leads to receptor downregulation. A) Representative flow cytometry result with Nef PROTACs FC-14369 (active) and FC-14379 (inactive). B) Active Nef PROTACs from the NanoBRET ubiquitination assay were screened for cell surface receptor rescue in triplicate. Bar height indicates the mean value ± SE; individual data points are also shown. The structures of the analogs with little to no activity in this assay (FC-13890, FC-14373, FC-14379, FC-14388) are shown in the Supplemental Information, Figure S2.

Figure 4.. Assessment of PROTAC-mediated Nef protein loss.

A) Flow cytometry of Nef-eGFP protein loss. CEM/Nef-eGFP cells were treated with doxycycline to induce expression of Nef-eGFP under conditions that result in a moderate level of positive cells by flow cytometry (see Figure 3A). Triplicate cultures of cells were treated with the Nef PROTAC analogs indicated at a final concentration of 3 μM, and 24 h later the percent of cells showing loss of Nef-eGFP protein expression were calculated relative to the DMSO controls and are presented as the mean value ± SE; individual data points are also shown. B) Correlation analysis of cell-surface CD4 rescue vs. Nef-eGFP protein loss (red data points, left) and MHC-I rescue vs. Nef-eGFP protein loss (blue data points, right). CD4 rescue was best-fit by linear regression, while MHC-I rescue showed a plateau effect. C) Immunoblot analysis. Cells expressing Nef-eGFP were treated as in part A with the eight active PROTACs, and lysates were prepared 48 h later for immunoblot analysis with Nef and Actin antibodies. A representative blot is shown. D) Immunoblot analysis was performed in duplicate, and band intensities were quantified by LI-COR infrared imaging and used to calculate Nef to Actin protein expression ratios. The bar graph shows the mean value for each ratio along with the individual values. The structures of the analogs with little to no activity in this assay (FC-13890, FC-14373, FC-14379, FC-14388) are shown in the Supplemental Information, Figure S2.

Figure 5.. Structures of active Nef PROTACs.

The Nef-targeting moiety (red, upper left) provides two positions on the phenyl rings for linker attachment (x and y). CRBN ligands included analogs of thalidomide (green, center) and lenalidomide (blue, right) that were attached to the isoindoline ring at positions 4 and 5. Linker compositions are shown below adjacent to each analog number. The complete structure of FC-14367 is also provided.

Figure 6.. Representative SPR sensorgrams for Nef PROTACs.

The thalidomide-binding domain of Cereblon (CRBN-TBD) and full-length Nef (NL4-3 variant) were expressed in E. coli and purified to homogeneity. A) Each protein was immobilized on one channel of a carboxymethyl dextran biosensor chip, and the two Nef PROTAC analogs FC-12988 and FC-13182 (structures at top; Nef-binding moiety in red, CRBN-binding ligands thalidomide and lenalidomide are shown in green and blue, respectively) were injected over the range of concentrations shown in the upper left sensorgram. Protein-ligand interaction was followed for 90 s, followed by a 180 s dissociation phase. The resulting data were fit to a 1:1 Langmuir binding model, and K_D values were calculated from the resulting association and dissociation rate constants (K_D values are summarized in Table S2). Each concentration was tested in triplicate, and individual traces are shown with the data shown in color and the fitted curves superimposed in black. RU, SPR response units. Arrows indicate the point of injection and the switch to wash buffer for dissociation. B) Comparison of SPR profiles for interaction of active PROTAC analog FC-14369 with Nef_NL4-3 vs. inactive analog FC-13906. The inactive analog dissociates rapidly from Nef while the active analog remains bound. Additional examples are provided in Figure S5.

Figure 7.. PROTACs inhibit Nef-dependent enhancement of HIV-1 replication in primary cells.

A) Donor PBMCs were infected with wild-type HIV-1_NL4-3 (DMSO control), a Nef-defective mutant (ΔNef), or wild-type virus in the presence of the Nef PROTACs shown at a final concentration of 1 μM. Input virus was 2,500 pg HIV p24 Gag per well. Replication was assayed by p24 Gag AlphaLISA 4 days later. Six independent determinations were assayed for each condition, and the highest and lowest p24 values were removed. Each bar indicates the mean ± SE of the remaining values with the individual data points shown. The dotted line indicates the mean value for the ΔNef control. Statistical significance was determined by Student’s t test between the DMSO control and ΔNef as well as each PROTAC treatment group; **, p < 0.01; ***, p < 0.001; ns, not significant. B) Viability of uninfected PBMCs was determined with each PROTAC at 1 μM after 4 days using the Cell Titer Blue assay (n = 6 wells/condition). The dotted line indicates 100% viability based on the DMSO control.