Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel–Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.
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The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary information files. Mass spectrometry proteomics data (Fig. 2e and Extended Data Fig. 2f) are provided in Supplementary Data Set 1. The study report of PKs of SIM1 following single i.v. and s.c. administrations to mice provided by Chempartner Co., Ltd., is provided in Supplementary Data Set 2. The study report of BROMOscan profiling service data (Fig. 5a) is provided as Supplementary Data Set 3. Source data are provided with this paper.
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We thank A. Bond for the synthesis of ARV-771, G. Liu, Q. Gu, N. Li, C. Ji, S. Liao, C. Wang, H. Li and B. Guan at ChemPartner for in vivo pharmacokinetic studies and W. Farnaby and T. Kirkland for discussions and critical reading of the manuscript. Research reported in this publication was supported by the European Research Council (ERC, starting grant ERC-2012-StG-311460 DrugE3CRLs to A.C.), the Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under grant agreement no. 875510 (EUbOPEN project) and Ono Pharma (visiting scientist fund to S.I.). K.-H. C. was funded by a Marie Skłodowska-Curie Actions Individual Fellowship from the European Commission (H2020-MSCA-IF-2014-655516). C.C. was funded by a PhD studentship from the UK Medical Research Council (MRC) under the doctoral training programme in Quantitative and Inter-disciplinary approaches to biomedical science (QI Biomed) (MR/N0123735/1). Biophysics and drug discovery activities were supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). K.M.R., S.D.M., N.M., M.U. and D.L.D. are funded by Promega Corporation.
The authors declare the following competing financial interest(s): the Ciulli laboratory receives or has received sponsored research support from Almirall, Amphista therapeutics, Boehringer Ingelheim, Eisai, Nurix therapeutics and Ono Pharmaceuticals. A.C. is a scientific founder, shareholder and consultant of Amphista therapeutics, a company that is developing targeted protein degradation therapeutic platforms. Promega Corporation is the commercial owner by assignment of patents of the HaloTag, NanoLuc, NanoBRET target engagement and HiBiT technologies and their applications, and K.M.R., S.D.M., N.M., M.U. and D.L.D. are employees of Promega Corporation. S.I. is an employee of Ono Pharmaceuticals. S.J.H. and A.T. are employees of Amphista therapeutics. K.-H.C. is an employee of GlaxoSmithKline.
Peer review information Nature Chemical Biology thanks Stefan Knapp and other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a) Inspection of tertiary complex crystal structures BRD4BD1:Bi-BET:BRD4BD1 (PDB: 5ad3) shows the bivalent inhibitor buried inside the protein interface, suggesting derivatization would impair the binding mode. b) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD4 HEK293 cells following treatment with 1 μM compounds or DMSO. Luminescence (RLU) was continuously monitored in 5 min intervals over a 24 h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. c) Immunoblot of BRD2, BRD3, BRD4 levels in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 and MZ1 for 4 h. Quantification of BET protein levels was done relative to DMSO control and plotted in Fig. 1e along with calculated DC50 values. The blot shown is representative of two independent experiments. Full blots are supplied as Source Data Files. d) Cell viability of A549 lung carcinoma cell line or HL-60 AML cell line following treatment with PROTACs or DMSO for 48 h in three replicates for each concentration point.
a) Identical live cell degradation SIM1 dose response graphs presented in Fig. 2a (right) of luminescence (RLU) plotted against time with a zoom-in of initial degradation within the first 3 h. b) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with serially diluted ARV-771. Luminescence (RLU) was continuously monitored in 5–15 min intervals over a 24 h time period and fractional RLU was determined by normalization to DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates. c) Comparison of BRD3 and BRD4 degradation rates, degradation maximum (Dmax), and Dmax50 values from kinetic profiles of SIM1 (Fig. 2a), ARV-771 (Extended Data Fig. 2b), and the previously determined MZ1 (Riching et.al. ACS Chem. Biol. 2018). d) NanoBRET ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 100 nM SIM1 or MZ1 or DMSO treatment in quadruplicate. Values are expressed as fold increase over DMSO control. e) Quantification of representative proteins of mass spectrometry proteomics on Fig. 2e. Data are presented as mean values with error bars representing the SD from triplicates. All t-tests performed were two-tailed assuming equal variances. Exact P values: SIM1 (BRD2 P = 0.00001, BRD3 P = 0.00024, BRD4 P = 0.00004, MYC P = 0.00072, HMOX1 P = 0.00082); cis-SIM1 (BRD2 P = 0.45, BRD3 P = 0.11, BRD4 P = 0.05, MYC P = 0.01, HMOX1 P = 0.04).
Extended Data Fig. 3 SIM1 shows potent Myc-driven activity in BET-sensitive AML and prostate cancer lines.
a) Loss in CRISPR cMyc-HiBiT protein levels (left quadrant) and correlative cell viability (right quadrant) in MV4;11 cells treated with 3 nM, 10 nM, 50 nM and 100 nM concentration of the indicated compounds. Luminescence and cell viability by CellTiter-Glo were measured at various time points over 24 h and normalized to the DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) (upper graphs) Quantified expression levels of endogenous BRD3 and BRD4 in 22Rv1 prostate cancer cell line treated with compounds for 4 h. Curves are a best fit of means from two biologically independent experiments. (lower image) Representative images of Western blots for Fig. 3b and upper graphs. Full blots are supplied as Source Data Files. c) Immunoblots of endogenous BRD2, BRD3 and BRD4 in 22Rv1 cells treated with SIM1 (100 nM) or cis-SIM1 (100 nM) with/without the addition of proteasome inhibitor (MG132, 3μM) or VHL inhibitor (VH298, 10μM) for 4 h. Blot is representative from two biologically independent experiments. Full blots are supplied as Source Data Files.
a) Immunoblot of PARP-cleavage in 22Rv1 cells with SIM1 or cis-SIM1 at 10 nM for the indicated time point with/without the addition of caspase inhibitor (QVD-OPh, 20 μM) or necroptosis inhibitor (Necrostatin-1, 20 μM). b) Immunoblot of PARP-cleavage in 22Rv1 cells with MT1 (left) or MZ1 (right) at 10 nM or 1 μM for the indicated time point with/without the addition of caspase inhibitor (QVD-OPh, 20 μM) or necroptosis inhibitor (Necrostatin-1, 20 μM). Full blots for panels (a-b) are supplied as Source Data Files. c) Caspase-Glo 3/7 assays in 22Rv1 cells treated in presence of BET degrader or inhibitor either in the absence (white bars) or presence of VHL inhibitor VH298 (orange bars) or caspase inhibitor QVD-OPh (red bars) for 24 h. Data are presented as mean values with error bars representing the SD from three biologically independent experiments. SIM1, cis-SIM1 and MT1 were used at 100 nM. MZ1 and ARV-771 were used at 1 μM. VH298 was used at 10 μM. QvD-OPh was used at 20 μM.
Images represent raw data-containing dot plots generated in FlowJo software by analysing 10000 events per condition. Staining for viability (DAPI) and phosphatidyl serine (FITC:Apotracker Green) is shown on the x-axis and y-axis, respectively. Different quadrants represent differently stained population of cells: Q1 (DAPI-/FITC + ), Q2 (DAPI + /FITC + ), Q3 (DAPI + /FITC-), Q4 (DAPI-/FITC-). Percentage of events per quadrant is denoted on individual graphs. Data shown are representative of n = 3 independent experiments. Gating was performed on a DMSO control and applied unchanged to other conditions, as shown in Supplementary Fig. 1.
a) Size exclusion chromatography of complex formation after incubation of SIM1 (red, 25 μM), MZ1 (orange, 25 μM), MT1 (green, 25 μM) or DMSO (cyan, 25 μM) with 25 μM BRD4 BD1-BD2 tandem protein (left panel: N433F mutant, right panel: N140F mutant with VCB protein). Intensity of peaks is absorbance 280 nM. b) NanoBRET target engagement assays with CRISPR HiBiT-BRD4 HEK293 cells stably expressing LgBiT performed in permeabilized and live cell formats. Cells were treated with a fluorescent BET tracer, then incubated with the indicated compounds at various concentrations to measure competitive displacement. IC50 values for each compound are shown for both permeabilized and live cells. Data are presented as mean values with error bars representing the SD of technical quadruplicates. c) NanoBRET ternary complex kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-VHL following 100 nM SIM1 or MZ1 or DMSO treatment. Values are expressed as fold increase over DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates.
a) Representative SPR sensorgrams for SIM1 (binary) and SIM1:BET tandem complexes (ternary) binding to immobilized VCB. Sensorgrams shown are for multi-cycle kinetic (MCK) experiments as representative of at least two independent experiments. Data were fitted globally to a 1:1 Langmuir model including a component for mass transfer effects. Fitted SPR parameters are in Supplementary Table 1. b) Live cell kinetic residence time experiments with BRD2 and BRD4 as measured by NanoBRET target engagement. CRISPR HiBiT-BRD2 or HiBiT-BRD4 cells were incubated with each of the indicated compounds in triplicate at their pre-determined EC80 values followed by addition of a competitive fluorescently-labeled BET tracer. NanoBRET was measured and is plotted as fractional occupancy over time. From these graphs, residence time rates (Kobs (h−1) and half-life (t½ (h)) were determined and are shown. Data are presented as mean values with error bars representing the SD of technical triplicates.
Extended Data Fig. 8 Cellular degradation and mechanistic analysis of CRBN-based trivalent degrader SIM4.
a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM4 over concentration range of 1nM-1mM. Luminescence (RLU) was continuously monitored over a 20 h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. (b,c) NanoBRET live cell assay of HiBiT-BET HEK293 cells expressing LgBiT with either HaloTag-CRBN for ternary complex studies (b) or HaloTag-Ubiquitin (c) and treated with DMSO, 0.1 mM SIM4, or 1 mM SIM4 treatment for 2.5 h. NanoBRET ratios were calculated at each time point and are graphed as fold increase over DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates. SIM4 shows only partial degradation (Dmax between 40–60%) for all BET family members. While ternary complex is observed with CRBN for BRD2, BRD3, and BRD4, minimal increase in ubiquitination is observed for all after treatment with SIM4.
Supplementary Fig. 1, Supplementary Tables 1 and 2, Supplementary Note (synthetic procedures) and NMR spectra.
Supplementary data set: proteomic analysis of relative protein abundance in MV4;11 cells.
Supplementary data set: pharmacokinetics of SIM1 following single i.v. and s.c. administrations to male C57BL/6 mice.
BROMOscan profiling service report and data.
Unprocessed western blots and/or gels for Fig. 1d,i.
Unprocessed western blots and/or gels for Fig. 3d and Extended Data Fig. 4a,b.
Unprocessed western blots and/or gels for Extended Data Fig. 1b.
Unprocessed western blots and/or gels for Extended Data Fig. 3b,c.
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Imaide, S., Riching, K.M., Makukhin, N. et al. Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity. Nat Chem Biol 17, 1157–1167 (2021). https://doi.org/10.1038/s41589-021-00878-4