Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Design and in vitro activity of TAPTAC1 (A) A chimera composed of a stapled p53 peptide and a small molecule BET protein inhibitor was designed to achieve triple-action targeting of HDM2, HDMX, and BET proteins to maximally restore p53 while hijacking HDM2 to degrade an oncogenic driver (BET proteins) instead of a tumor suppressor (p53). (B) Chemical composition of TAPTAC1, which links the BET inhibitor JQ1 to the stapled p53 peptide via a lysine-βAla moiety installed at position 25 of the p53 transactivation helix. (C) MD simulations demonstrate the assembly of a ternary complex between TAPTAC1 and the respective JQ1- and p53-binding domains of BRD4 and HDM2. (D) TAPTAC1 effectively generated ternary complexes (green) between the BD1 domain of BRD4 and HDM2 (left) and HDMX (right). Control elution profiles are shown for the individual proteins alone, including BRD4 BD1 (cyan), HDM2 (red), and HDMX (orange), and their combinations, including BRD4 BD1 and HDM2 (purple) and BRD4 BD1 and HDMX (brown). Each SEC experiment was repeated twice using independent preparations of proteins with similar results. (E) An in vitro ubiquitylation assay demonstrated the natural selectivity of HDM2 for p53, as evidenced by time-dependent laddering of p53 but not BRD4 BD1-BD2 (left 4 lanes). In the presence of TAPTAC1, the primary target of HDM2 is switched from p53 to BRD4 BD1-BD2 , which exhibits newfound laddering at the expense of p53 (right 4 lanes). Ubiquitylation assays were repeated three times with independent preparations of proteins and reagents with similar results. See also and .

Article Snippet: The human BRD4 BD1 domain (residues 48–168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg/mL kanamycin.

Techniques: In Vitro, Activity Assay, Binding Assay, Generated, Control, Ubiquitin Assay

Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Potent and selective cytotoxicity of TAPTAC1 correlates with degradation of BET proteins and reactivation of the p53 pathway (A) To assess the relative potency and selectivity of TAPTAC1, we compared its anti-cancer activity in culture with A1874, a 2-in-1 PROTAC comprising the selective HDM2 inhibitor RG7388 and JQ1, and the TAPTAC1 F19A point mutant, which abrogates interaction of the stapled p53 peptide component of the chimera with HDM2 and HDMX. (B–D) In SJSA-1 cells that have genetic amplification of HDM2 but little to no HDMX expression, TAPTAC1 exhibits marginally increased potency compared to A1874, as assessed by viability assay (B). In SJSA-X cells, engineered to express HDMX, the dual-targeting capability of TAPTAC1 results in markedly enhanced potency compared to A1874 (C). In Saos-2 cells, which lack p53, no p53/HDM2/HDMX-based activity or selectivity is evident, consistent with the compounds functioning similarly based on residual BET inhibitor activity alone (D). In each cell line, F19A point mutagenesis likewise abrogates the p53-dependent activity of TAPTAC1, further highlighting its specificity of action. Data are mean ± SEM for experiments performed in technical quadruplicate and repeated three times using independent cultures with similar results. (E) Upon treatment of SJSA-X cells with 100 nM TAPTAC1, we observed prompt degradation of BRD4 within 8 h, coinciding with time-dependent upregulation of p53, which peaked at 12 h and triggered both a surge in p21 and counter-elevation of HDM2 and HDMX by 24 h. The reduction of p53 levels observed between 12 and 24 h is consistent with the characteristic negative feedback loop of the p53 pathway. Time-dependent western blot analyses were performed twice using independent SJSA-X cell cultures and compound treatment. NT, no treatment. (F) Quantitative proteomics revealed that TAPTAC1 treatment (1 μM, 24 h) of SJSA-X cells caused a striking reduction of BET protein levels (cyan, e.g., BRD2-4) and marked upregulation of p53 pathway proteins (red, e.g., p53 [TP53], p21 [CDKN1A], HDM2 [MDM2], and HDMX [MDM4]). Quantitative proteomic analysis was performed using three biological replicates representing independent cultures and treatment. ∗, isoform. See also , , and .

Article Snippet: The human BRD4 BD1 domain (residues 48–168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg/mL kanamycin.

Techniques: Activity Assay, Mutagenesis, Amplification, Expressing, Viability Assay, Western Blot, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Pharmacologic profile and therapeutic efficacy of TAPTAC1 in a mouse model of osteosarcoma (A) TAPTAC1 was incubated in mouse or human plasma, and the relative amount of intact compound was measured over time by mass spectrometry, revealing notable compound stability. Compound levels were tracked over time (6 time points each) in an individual sample of mouse or human plasma. (B and C) Mice ( n = 3/arm) received 3 mg/kg of TAPTAC1 by intravenous (IV) or intraperitoneal (IP) injection (B) or 3 or 10 mg/kg TAPTAC1 by IV injection (C), followed by serial blood withdrawal for mass spectrometry quantitation of compound. Calculated parameters for IP (3 mg/kg) and IV (3 mg/kg and 10 mg/kg) administration, respectively, included T 1/2 5.33, 5.01, and 4.71 h; C max 0.94, 2.1, and 31.2 μM; and AUC last 8.3, 10.3, and 61.7 μM∗hr. Plotted pharmacokinetic data are mean ± SEM for experiments performed in three mice per arm. (D) Treatment of NSG mice bearing SJSA-X osteosarcoma tumors (mean tumor volume ±SD of 401 ± 53mm 3 on treatment day 1) with 10 mg/kg TAPTAC1 IV daily (qd) resulted in marked shrinkage of tumors as compared to the unabetted tumor growth observed in vehicle-treated mice. Plotted data are mean tumor volume ±SEM as measured daily ( n = 5/arm). Statistical analysis using a longitudinal mixed-effects model revealed a significant difference in tumor volume trajectories between the treated and vehicle groups ( p < 0.001). (E) Photographs of tumors removed postmortem on treatment day 7 from each of three mice treated with either vehicle or TAPTAC1 (10 mg/kg/day IV) revealed the striking anti-tumor effect of TAPTAC1. (F) Quantitative proteomics (TMTpro 18-plex) of the tumor specimens demonstrated marked downregulation of BRD4 and persistent upregulation of HDMX (MDM4), in TAPTAC1- vs. vehicle-treated mice at day 7, in addition to changes that reflect replacement of tumor with host connective tissue. Quantitative proteomic analysis was performed using three biological replicates ( n = 3 tumor specimens) per treatment arm. (G) The in vivo efficacy experiment was repeated with reduced dosing to 3.0, 1.0, and 0.3 mg/kg/day IV (mean tumor volume ±SD of 216 ± 47 mm 3 on treatment day 1) and demonstrated dose-responsive anti-tumor activity and associated prolongation of survival. Data are mean tumor volume ±SEM as measured daily ( n = 5/arm). Statistical analysis using a longitudinal mixed-effects model revealed a significant difference in tumor volume trajectories between the 3 mg/kg dosing arm and vehicle group (vehicle vs. TAPTAC1 at: 0.3 mg/kg, p = 0.651; 1 mg/kg, p = 0.072; 3 mg/kg, p < 0.001). (H–J) Lowering the TAPTAC1 dosing interval to 3 mg/kg twice weekly (biw) also suppressed SJSA-X tumor growth relative to vehicle throughout the month-long treatment period (longitudinal mixed-effects model, vehicle vs. TAPTAC1 at 3 mg/kg BIW, p < 0.0001) (H), with no significant difference between the groups in animal weight trajectory ( p = 0.07) and marked prolongation of survival for TAPTAC1-treated mice (log rank test, p = 0.00033). The study was conducted with n = 6 vehicle-treated mice and n = 7 TAPTAC1-treated mice. See also , , and .

Article Snippet: The human BRD4 BD1 domain (residues 48–168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg/mL kanamycin.

Techniques: Drug discovery, Incubation, Clinical Proteomics, Mass Spectrometry, Injection, IV Injection, Quantitation Assay, Quantitative Proteomics, In Vivo, Activity Assay

Journal: Nature chemical biology

Article Title: Plasticity in binding confers selectivity in ligand induced protein degradation

doi: 10.1038/s41589-018-0055-y

Figure Lengend Snippet: (a) The chemical structure of dBET23 is depicted with the target-moiety in red, the linker in black and cyan, and the E3-moiety in blue. (b) Cartoon representation of DDB1ΔB-CRBN-dBET23-BRD4 BD1 : DDB1 highlighting domains BPA (red), BPC (orange) and DDB1-CTD (grey); CRBN with domains NTD (blue), HBD (cyan) and CTD (green); BRD4 BD1 (magenta). The Zn 2+ -ion is drawn as a grey sphere and dBET23 as sticks representation in yellow. The F O -F C map is shown as green mesh for dBET23 contoured at 3.0σ. (c) Superposition of DDB1ΔB-CRBN-dBET23-BRD4 BD1 with CRBN bound to lenalidomide (pdb: 4tz4) and BRD4 BD1 bound to JQ1-(S) (pdb: 3mxf). Surface representation for CRBN and BRD4 BD1 are shown in gray and magenta, respectively. dBET23 is shown in yellow, JQ1 in green, and thalidomide in cyan. (d) Side-chain interactions between BRD4 BD1 , CRBN, and dBET23. Residues of BRD4 BD1 mutated in this study are highlighted in cyan.

Article Snippet: His 6- TEV-BRD4 BD1 construct used for selenomethionine derivative was a gift from Sirano Dhe-Paganon (originally AddGene: 38942) and was expressed in B834-DE3 cells in SeMet Medium (Molecular Dimensions, MD12-500) following the manufacturer’s instructions.

Techniques:

Journal: Nature chemical biology

Article Title: Plasticity in binding confers selectivity in ligand induced protein degradation

doi: 10.1038/s41589-018-0055-y

Figure Lengend Snippet: (a) TR-FRET. dBET23 titrated to DDB1ΔB-CRBN SPY-BODIPY , Terbium-Streptavidin and various BRD4 BD1-biotin wild type and mutant proteins. The mean peak heights for dose response curves of three independent replicates are shown as dot-plot. Data in this figure is presented as means ± s.d. (n=3). (b) Competitive binding assay for dBET1 binding to DDB1ΔB-CRBN. Increasing concentrations of dBET1 titrated to preformed DDB1ΔB-CRBN-lenalidomide Atto565 complex in presence or absence of BRD4 BD1 or BRD4 BD2 . (c) As in b but using dBET6, (d) dBET23, or (e) dBET57. All data in this figure are independent replicates presented as means ± s.d. (n=3).

Article Snippet: His 6- TEV-BRD4 BD1 construct used for selenomethionine derivative was a gift from Sirano Dhe-Paganon (originally AddGene: 38942) and was expressed in B834-DE3 cells in SeMet Medium (Molecular Dimensions, MD12-500) following the manufacturer’s instructions.

Techniques: Mutagenesis, Competitive Binding Assay, Binding Assay

Journal: Nature chemical biology

Article Title: Plasticity in binding confers selectivity in ligand induced protein degradation

doi: 10.1038/s41589-018-0055-y

Figure Lengend Snippet: (a) Quantitative assessment of cellular degradation using a BRD4 BD1 -EGFP reporter assay. Cells stably expressing BRD4 BD1 -EGFP and mCherry were treated with increasing concentrations of dBET1, dBET6, dBET23, dBET55, dBET57, dBET70, MZ1 and lenalidomide and the EGFP and mCherry signals followed using flow cytometry analysis. (b) as in a but using cells that express a BRD4 BD2 -EGFP reporter along with mCherry. Data in a and b are EGFP and mCherry signals quantified using flow cytometry analysis. Data of independent cell culture replicates is presented as the means ± s.d. (n=3 for MZ1, dBET6, 23, 55; n=4 for dBET1, 57, 70; n=6 for lenalidomide).

Article Snippet: His 6- TEV-BRD4 BD1 construct used for selenomethionine derivative was a gift from Sirano Dhe-Paganon (originally AddGene: 38942) and was expressed in B834-DE3 cells in SeMet Medium (Molecular Dimensions, MD12-500) following the manufacturer’s instructions.

Techniques: Reporter Assay, Stable Transfection, Expressing, Flow Cytometry, Cell Culture

Journal: Nature chemical biology

Article Title: Plasticity in binding confers selectivity in ligand induced protein degradation

doi: 10.1038/s41589-018-0055-y

Figure Lengend Snippet: (a) TR-FRET. dBET23 titrated to BRD4 BD1-SPYCATCHER-BODIPY , Terbium-antiHis antibody and various His6-DDB1ΔB-CRBN wild type and His6-DDB1-CRBN mutant proteins. The mean peak heights for dose response curves of three independent replicates are shown as dot-plot. TR-FRET data in this figure is presented as means ± s.d. (b) surface representation of CRBN highlighting the residues involved in dBET23 mediated BRD4 BD1 binding in orange (residues Y59, L60, Q86, Q100, F102, H103, P104, D149, F150, G151, I152, I154, K156, P352, H353, E377, H378). CRBN interface residues mutated for biochemical assays are indicated. (c) TR-FRET. dBET23 titrated to DDB1ΔB-CRBN SPYCATCHER-BODIPY , Terbium-Streptavidin and various BRD4 BD1-biotin wild type and mutant proteins. The mean peak heights for dose response curves of three independent replicates are shown as dot-plot. TR-FRET data in this figure is presented as means ± s.d. (d) as in a but titrating dBET57. (e) surface representation of CRBN highlighting the BRD4 BD1 interacting residues for the dBET57 mediated recruitment in orange (residues: Q325, H353, Y355, H357, I371, G372, R373, E377, V388, Q390, C394, A395, S396, H397, T418, S420). CRBN interface residues mutated for biochemical assays are indicated. (f) as in b but titrating dBET57. (g) Cartoon representation of DDB1ΔB-CRBN-dBET57-BRD4 BD1 : DDB1 highlighting domains BPA (red), BPC (orange) and DDB1-CTD (grey); CRBN with domains NTD (blue), HBD (cyan) and CTD (green); BRD4 BD1 (magenta). The Zn 2+ -ion is drawn as a grey sphere. dBET57 was not modelled in this structure but instead superpositions of lenalidomide (from pdb: 5fqd) and JQ1 (from pdb: 3mxf) are shown in yellow sticks. (h) Superposition of CRBN and BRD4 BD1 for the dBET23 and dBET57 containing complexes. Superposition was carried out over the CRBN-CTD (residues 320 – 400). (i) The chemical structures of dBET57 is depicted with the target-moiety in red, the linker in black and cyan, and the E3-moiety in blue.

Article Snippet: His 6- TEV-BRD4 BD1 construct used for selenomethionine derivative was a gift from Sirano Dhe-Paganon (originally AddGene: 38942) and was expressed in B834-DE3 cells in SeMet Medium (Molecular Dimensions, MD12-500) following the manufacturer’s instructions.

Techniques: Mutagenesis, Binding Assay

Journal: Nature chemical biology

Article Title: Plasticity in binding confers selectivity in ligand induced protein degradation

doi: 10.1038/s41589-018-0055-y

Figure Lengend Snippet: (a) Cartoon representations for representative clusters obtained by k-means clustering of the top 200 global docking poses between CRBN (pdb: 4tz4) and BRD4 BD1 (pdb: 3mxf). (b) Histogram of the pairwise shortest distances for the top 200 docking poses. (c) Close-up view on the proximity of the JQ1 thiophene and lenalidomide that provided the rationale for synthesizing ZXH-2-147 and ZXH-3-26. Atoms used for calculation of the pairwise shortest distances between JQ1 and lenalidomide are highlighted in black circles.

Article Snippet: His 6- TEV-BRD4 BD1 construct used for selenomethionine derivative was a gift from Sirano Dhe-Paganon (originally AddGene: 38942) and was expressed in B834-DE3 cells in SeMet Medium (Molecular Dimensions, MD12-500) following the manufacturer’s instructions.

Techniques:

Journal: Nature chemical biology

Article Title: Plasticity in binding confers selectivity in ligand induced protein degradation

doi: 10.1038/s41589-018-0055-y

Figure Lengend Snippet: (a) Chemical structure of ZXH-3-26. (b) Quantitative assessment of cellular degradation using a EGFP/mCherry reporter assay. Cells stably expressing BRD4 BD1 -EGFP (or constructs harboring BRD2 BD1 , BRD2 BD2 , BRD3 BD1 , BRD3 BD2 , BRD4 BD2 ) and mCherry were treated with increasing concentrations of ZXH-3-26 and the EGFP and mCherry signals followed using flow cytometry analysis (c) As in b but for dBET6, MZ1 (d) , and dBET57 (e) . Data in b-e are representative experiments out of at least three experiments. (f) Cellular degradation of endogenous BRD4. HEK293T cells were treated with increasing concentrations of ZXH-3-26 or dBET6 for 5 hours, and protein levels assessed by western blot. (g) As in f but assessing the degradation of BRD2 and BRD3 by western blot. Experiments in f and g are representative of two independent experiments. Full scans for all western blots are provided in . (h) Scatter plot depicting the fold changes in relative abundance comparing 0.1 μM ZXH-3-26 to DMSO control treatment for 4 hours in MM.1s cells determined using quantitative proteomics. Negative log 10 false discovery rate adjusted P Values are shown on the x-axis and log2 fold changes on the y-axis. BRD4 is significantly downregulated with a log2 fold change of -1.99, and a FDR adjusted P value of 0.0018. Data shown are of three cell culture replicates measured in a single 10-plex TMT experiment (showing 6311 proteins quantified by > 3 unique peptides). P Values were derived from a moderated t -statistic using the limma package and corrected for multiple hypothesis testing using a Benjamini-Hochberg approach (see methods for details) .

Article Snippet: His 6- TEV-BRD4 BD1 construct used for selenomethionine derivative was a gift from Sirano Dhe-Paganon (originally AddGene: 38942) and was expressed in B834-DE3 cells in SeMet Medium (Molecular Dimensions, MD12-500) following the manufacturer’s instructions.

Techniques: Reporter Assay, Stable Transfection, Expressing, Construct, Flow Cytometry, Western Blot, Control, Quantitative Proteomics, Cell Culture, Derivative Assay

Journal: bioRxiv

Article Title: Suramin potently inhibits binding of the mammalian high mobility group protein AT-hook 2 to DNA

doi: 10.1101/838656

Figure Lengend Snippet: The discovery of suramin as a specific and potent inhibitor of HMGA-DNA interactions. (A) The AlphaScreen Primay assay with IC 50 of 2.6 μM. (B) The TR-FRET LANCE secondary screen. (C) The couterscreen assay using the BRD4 AlphaScreen Assay. (D) The cytotoxicity assay. All assays were described in Materials and Methods. The standard deviation was calculated according to three independent experiments. The curve represents the best fit of a four parameter logistic that was determined by nonlinear regression. Data points represent mean ±SEM.

Article Snippet: His-tagged BRD4-BD1 was purchased from BPS Bioscience (#31042).

Techniques: Amplified Luminescent Proximity Homogenous Assay, Cytotoxicity Assay, Standard Deviation