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.2021 Oct 28;64(20):15477-15502.
doi: 10.1021/acs.jmedchem.1c01532. Epub 2021 Oct 15.

Development of BromoTag: A "Bump-and-Hole"-PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

Affiliations

Development of BromoTag: A "Bump-and-Hole"-PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

Adam G Bond et al. J Med Chem..

Erratum in

Abstract

Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Here, we design and develop the BromoTag, a new inducible degron system comprising a Brd4 bromodomain L387A variant as a degron tag that allows direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. We describe extensive optimization and structure-activity relationships of our bump-and-hole-PROTACs using a CRISPR knock-in cell line expressing model target BromoTag-Brd2 at endogenous levels. Collectively, our cellular and mechanistic data qualifies bumped PROTAC AGB1 as a potent, fast, and selective degrader of BromoTagged proteins, with a favorable pharmacokinetic profile in mice. The BromoTag adds to the arsenal of chemical genetic degradation tools allowing us to manipulate protein levels to interrogate the biological function and therapeutic potential in cells andin vivo.

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Conflict of interest statement

The authors declare the following competing financial interest(s): The University of Dundee has filed a patent application (GB2113656.9) related to the BromoTag technology. The A.C. laboratory receives or has received sponsored research support from Almirall, Amphista Therapeutics, Boehringer Ingelheim, Eisai Co., Nurix, and Ono Pharmaceuticals. A.C. is scientific founder, shareholder and consultant of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms.

Figures

Figure 1
Figure 1
(A) Pan-selective BET degraders, MZ1 andARV-771. (B) Pan-selectiveBET inhibitors, (+)-JQ1 and I-BET762 (top). Allele-specific bumpedBET inhibitors, ME, ET, 9-ME-1, and 9-ET-1 (bottom). (C) Tailoringthe “bump-and-hole” approach to BET bromodomains toproduce a high-affinity selective pairing that can be utilized asa degron system. (D) Conceptualization of the BromoTag degron approach.
Figure 2
Figure 2
Design anddevelopment of a heterozygous knock-in BromoTag-Brd2HEK293 cell line. (A) Design of the knock-in construct used in thedevelopment of the CRISPR construct. (B) FACS single cell sort ofHEK293 cells based on GFP expression. Successive single cells weresorted into individual wells of a 96-well plate. (C) Junction PCRusing genomic DNA of an expanded GFP-expressing clone paired againstparental HEK293. (D) Western blot demonstrating the selectivity ofthe polyclonal Brd4BD2L387A. antibody.
Figure 3
Figure 3
(A) Ternary complex betweenBrd4BD2 (green, cartoon/surfacerepresentation), MZ1 (1, stick, gray carbons), and VCB(VHL: blue; elongin C: pink; elongin B—pale orange; and cartoon/surfacerepresentations). Leu387 (stick, green) is indicated by an arrow (PDBcode: 5T35).Alignment of Brd4BD2 (pale green, cartoon representation, 5T35) with (B) Brd2BD2 L383A (orange, cartoon representation, 4QEW) and (C) Brd2BD2 L383V (yellow, cartoon representation, 5O3C), co-crystallizedwith MZ1 (1, stick, gray carbons), ET (6, stick, pink carbons), and 9-ME-1 (7, stick, blue carbons),respectively. Brd4BD2 W.T. Leu387 (stick, pale greencarbons) and mutants Brd2BD2 L383A Ala383 (stick,orange carbons) and Brd2BD2 L383V Val383 (stick, yellowcarbons) are highlighted.
Scheme 1
Scheme 1. Synthesis of I-BET762-Based B&H–PROTACs and Non-bumped Control Compound
Reaction conditions: (a) 10%Pd/C, H2, MeOH, r.t., and 3 h; (b)10, HATU,DIPEA, DCM, r.t., and 18 h; and (c) bumped I-BET acid11,12, or13; HATU; HOAt; DIPEA; DMF; r.t.;and 18 h. * indicates relative configuration at the specified stereogeniccenters in the molecule.
Figure 4
Figure 4
First-generation IBET-762-based B&H–PROTACsare inactiveagainst BromoTag-Brd2 due to the proposed steric clash in the MZ1-liketernary complex. (A) Western blot data for BET protein levels afterthe treatment of PROTAC over 6 h in heterozygous BromoTag-Brd2 HEK293cells. (B) Alignment of the ternary complex between Brd4BD2 (green, surface representation), MZ1 (1, stick, graycarbons), and VHL (cyan, surface representation) (PDB code: 5T35), with ET (6, 8-OMe, stick, pink carbons, 4QEW) and 9-ME-1 (7, 9-OMe, stick,blue carbons, 5O3C) to show the potential clash with VHL by the bulkier 8/9-methoxyphenylgroup. His110 is highlighted (stick, cyan carbons).
Scheme 2
Scheme 2. Synthesis of Racemic Bumped JQ1 Ligands
Reaction conditions: (a) (i)SOCl2, DCM, reflux, and 2 h; (ii)27, CHCl3, reflux, and 1 h; (iii) TEA, reflux, and 16 h; and (iv) AcOH,1,2-DCE, 80 °C, and 1 h; (b) (i) KOtBu, THF,−78 to −10 °C, and 30 min; (ii) (EtO)2P(O)Cl, −78 to −10 °C, and 45 min; (iii) AcNHNH2, r.t., and 1 h; (iv)n-BuOH, 90 °C,and 1 h; (c) (i) KHMDS, THF, −78 °C, and 1 h; (ii) MeI/EtI,−78 °C to r.t., and 16 h, and (iii) HPLC separation; (d)(i) NaOMe, MeOH, 120 °C m.w., and 40 min and (ii) HPLC separation;and (e) LiOH, THF/H2O 4:1,30a, r.t., 48–72h,30b, 45 °C, and 1 wk. * indicates relative configurationat the specified stereogenic centers in the molecule.
Scheme 3
Scheme 3. Conjugation of Linkers to VHL Ligands
Reaction conditions:(a) HATU,DIPEA, DMF, r.t., and 2 h and (b) TBAF, THF, r.t., and 6 h.
Scheme 4
Scheme 4. Synthesis of JQ1-Based B&H–PROTACs as Mixtures of Two Diastereomers
Reaction conditions: (a) 10%Pd/C, H2, MeOH, r.t., and 3 h; (b) bumped JQ1 acid (32 or33), COMU, DIPEA, THF, r.t., and 4 h; and(c) bumped JQ1 acid (32 or33), EDC·HCl,DMAP, THF, r.t., and 16 h. * indicates relative configuration at thespecified stereogenic centers in the molecule.
Figure 5
Figure 5
Biologicalevaluation of second-generation B&H–PROTACsin BromoTag-Brd2 HEK293 cells. Western blot data for BET protein levelsmonitored from 10 μM to 1 nM compound treatment over 6 h inheterozygous BromoTag-Brd2 HEK293 cells. Bands are normalized to tubulinand negative control (cis-MZ1) to derive DC50 values that enable the rank order of each PROTAC.
Scheme 5
Scheme 5. Synthesis of Enantiomerically Pure AGB1, AGB2, and AGB3
Reaction conditions: (a) (i)SOCl2, DCM, r.t., and 3 h; (ii)41 or44, DCM, r.t., and 16 h; (b) 10% Pd/C, H2, MeOH,r.t., and 3 h; and (c)45, COMU, DIPEA, DMF, r.t., and2 h.
Figure 6
Figure 6
Biological evaluationof AGB1 (46), AGB2 (47), and AGB3 (48) in BromoTag-Brd2 HEK293 cells. (A)Western blot data for BET protein levels monitored from 10 μMto 1 nM compound treatment over 6 h in heterozygous BromoTag-Brd2HEK293 cells. (B) Time course western blot data of Brd2 levels inheterozygous BromoTag-Brd2 HEK293 cells upon 500 nM treatment of46 and47 and 1 μM treatment of48 over 36 h. (C,D) Plots to calculate (C) DC50 and (D)t1/2 values for compoundsenabling determination that AGB1 is the best choice for further validation.Western blots from (A,B) were normalized to tubulin and compared toa vehicle control (DMSO) to derive pDC50 ort1/2 values that enable rank order of each PROTAC.
Figure 7
Figure 7
FPof B&H–PROTAC binary and ternary complex binding.Binary and ternary complex formation FP data for46 (A),47 (B), and48 (C) to VHL alone (black solidline) or preincubated with Brd4BD2 L387A to VHL (coloreddashed line), respectively. Error bars andKd values are mean (±S.E.M.) fromN =4 for binary and ternary binding to VHL. The left shift between thebinary and ternary data indicates positive cooperativity. Cooperativity(α) calculated as a ratio ofKdbinary/Kdternary.
Figure 8
Figure 8
Cellularmechanistic characterization of AGB1 (46)degradation activity. (A) Western blot illustrating theon-target degradation activity of46 is dependent on theactivity of CRL2VHL and proteasome and on BromoTag targetengagement. BromoTag-Brd2 HEK293 cells were treated with 200 nM46 (3 h) following pretreatment (1 h) with the proteasomeinhibitor MG132, neddylation inhibitor MLN4924, VHL inhibitor VH298,or BromoTag inhibitor ET-JQ1-OMe or DMSO vehicle. (B) Western blotsdemonstrating the recovery of BromoTag-Brd2 post-removal of 200 nM46 after a 3 h treatment in heterozygous BromoTag-Brd2 HEK293cells. Control experiments for no-wash and vehicle treatments areincluded. Bands are normalized to tubulin protein levels and comparedto a vehicle control (DMSO) to quantify the final protein levels ofBromoTag-Brd2. (C) Effect on antiproliferation of46 comparedto MZ1 and non-degrader controls52 andcis-MZ1. Staurosporine was used as a positive control for cytotoxicity.MV-4-11, 22Rv1, and HEK293 cells were treated with varying concentrationsof compound, and after 24, 48, and 48 h, respectively, the cells weresubjected to the Promega CellTiter-Glo cell viability assay. The pEC50 values (±S.E.M) are mean fromN =2 for MV-4-11 and 22Rv1 cells andN = 3 for HEK293cells from data normalized from vehicle control (DMSO).
Scheme 6
Scheme 6. Synthesis of Negative Controlcis-AGB1 (52)
Reaction conditions: (a)37, COMU, DIPEA, DMF, r.t.,and 2 h; (b) TBAF, THF, r.t.,and 6 h; and (c) (i)45, SOCl2, DCM, r.t.,and 3 h and (ii)51, DCM, r.t., and 16 h.
Figure 9
Figure 9
Proteomics of AGB1 (46) andcis-AGB1(52) treated heterozygous BromoTag-Brd2 HEK293 cells.Scatterplot depicting the effect of46 (blue) and52 (red) treatment on the proteome of heterozygous BromoTag-Brd2HEK293 cells treated with 1 μM of compound for 2 h. Brd2 expressionis highlighted for both treatment conditions. The data plotted islog2 of the normalized fold change in abundance against−log10 of the p value per protein identified fromTMT mass spectrometry analysis produced from three independent experiments.
Figure 10
Figure 10
Plasma stability andin vivo PK studies of AGB1(46) in mice. (A) Percentage of46 remainingafter 0, 5, 15, 30, 45, and 60 min in mouse plasma at 37 °C,normalized to 0 min time point, with two independent repeats per timepoint. (B) Male C57BL/6 mice were treated with a single 5 mg/kg doseof46 by either IV (black dots) or SC (hollow squares)injection, and the blood plasma concentration of46 wasmeasured at seven time points. Data is mean (±S.D.) from threeindependent repeats at each time point. The red dashed line indicatesthe DC50, 6h of46 for degrading BromoTag-Brd2.
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