Journal: Advanced Science

Article Title: A Temporal PROTAC Cocktail‐Mediated Sequential Degradation of AURKA Abrogates Acute Myeloid Leukemia Stem Cells

doi: 10.1002/advs.202104823

Figure Lengend Snippet: Design and characterization of different E3 ubiquitin ligase‐based AURKA PROTACs. A) Crystal structure of AURKA in complex with MLN8054 (PDB code 2×81). The lysine residues on the surface of AURKA are highlighted. B) Chemical structures of AURKA PROTAC based on MLN8237 and the ligands of CRBN, VHL, and cIAP. MW, molecular weight (Daltons). C) SPR sensorgrams and KD values of MLN8237 and AURKA PROTACs binding to the AURKA protein. D) Dual‐colored fluorescence SPPIER (DF‐SPPIER) imaging of ternary complex formation in HEK293T cells with PROTACs and E3 ligands induction (500 × 10 −9 m ) for 3 h. Fluorescence histogram of the line across the cells (normalized fluo., normalized fluorescence intensity) and the log10 normalized sum of pixel fluorescence intensity (normalized SPPIER, log 10 (intensity + 1)) of yellow droplets in each cell are shown on the right. Scale bar: 5 µm. E) Degradation of endogenous AURKA in KG1A cells following 6 h treatment with the indicated concentration of PROTACs. F) Degradation of endogenous AURKA in KG1A cells following 6 h treatment with the indicated PROTACs (500 × 10 −9 m ) and E3 ligase ligands. G) Degradation of endogenous AURKA in KG1A cells with or without doxycycline (DOX, 0.5 µg mL −1 ) treatment for 72 h following 6 h treatment with the indicated PROTACs (500 × 10 −9 m ). H) TMT‐based quantitative proteomics after treatment with dAurA383 (500 × 10 −9 m ), dAurA450 (500 × 10 −9 m ) or the DMSO Vehicle for 6 h in KG1A cells. The differentially expressed proteins are presented in the volcano plot. I. KG1A cells were synchronized at the G1/S boundary by a double thymidine method, released into fresh media, and harvested at the indicated times (T/T release). The cell cycle profile was assayed by FACS with propidium iodide (PI) staining. Ctrl, proliferating KG1A cells under normal cell culture condition were used as control. J) The protein expression levels of the indicated proteins after T/T release were measured by Western blot. K) Schematic depiction of the relative protein levels of AURKA, CRBN, cIAP1, and VHL in KG1A cells. Statistics, significance: one‐way ANOVA with Bonferroni correction (D); ns, not significant; *** P < 0.001.

Article Snippet: In brief, DMSO, AURKA PROTACs, and ATRA (A9120, Solarbio) treated leukemia cells from the in vitro and in vivo assays were collected, counted, and washed with PBS.

Techniques: Ubiquitin Proteomics, Molecular Weight, Binding Assay, Fluorescence, Imaging, Concentration Assay, Quantitative Proteomics, Staining, Cell Culture, Control, Expressing, Western Blot

Journal: Advanced Science

Article Title: A Temporal PROTAC Cocktail‐Mediated Sequential Degradation of AURKA Abrogates Acute Myeloid Leukemia Stem Cells

doi: 10.1002/advs.202104823

Figure Lengend Snippet: Characterization of cellular response to PROTACs in AML cells. A) KG1A and Kasumi‐1 cells were stained with a carboxyfluorescein succinimidyl amino ester (CFSE) probe and cultured with AURKA PROTACs (1 × 10 −6 m ) for 72 h. CFSE fluorescence was analyzed by flow cytometry. MFI, mean fluorescence intensity. B) KG1A cells were treated with AURKA PROTACs (1 × 10 −6 m ) for 48 h. The cell cycle profile was assayed by FACS with propidium iodide (PI) staining. The cell cycle phase distribution was analyzed by FlowJo software. C) A heatmap of the relative normalized abundance of proteins in TMT‐based quantitative proteomic assays. D) TMT‐based quantitative proteomics after treatment with PROTACs (500 × 10 −9 m ) or the DMSO Vehicle for 6 h in KG1A cells. The top 100 decreased proteins were subjected to g:Profiler to perform gene ontology (GO) analysis. E) Gene set enrichment analysis (GSEA) of RNA‐seq data from KG1A cells treated with AURKA PROTACs (1 × 10 −6 m ) for 48 h. F) KG1A cells were treated with AURKA PROTACs (1 × 10 −6 m ) or ATRA (0.6 × 10 −6 m ) for 48 h. Cell surface CD34 expression was analyzed by FACS. MFI, mean fluorescence intensity. G) KG1A cells were arrested with nocodazole (0.1 µg mL −1 ) for 16 h followed by PROTACs or MLN8237 treatment with indicated concentration for 6 h. AURKA localization were detected by immunofluorescence. Scale bar: 5 µm. H) KG1A cells were arrested with nocodazole (0.1 µg mL −1 ) for 16 h followed by DMSO, PROTACs (1 × 10 −6 m ) or MLN8237 (50 × 10 −9 m ) treatment for indicated times. The expression of AURKA and related proteins were detected. Statistics, significance: one‐way ANOVA with Bonferroni correction (A,F); ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: In brief, DMSO, AURKA PROTACs, and ATRA (A9120, Solarbio) treated leukemia cells from the in vitro and in vivo assays were collected, counted, and washed with PBS.

Techniques: Staining, Cell Culture, Fluorescence, Flow Cytometry, Software, Quantitative Proteomics, RNA Sequencing, Expressing, Concentration Assay, Immunofluorescence

Journal: Advanced Science

Article Title: A Temporal PROTAC Cocktail‐Mediated Sequential Degradation of AURKA Abrogates Acute Myeloid Leukemia Stem Cells

doi: 10.1002/advs.202104823

Figure Lengend Snippet: An AURKA PROTAC cocktail relieves the hook effect. A) Scheme depicting the strategy to combine dAurA383 and dAurA450 to relieve the hook effect. B) Degradation of endogenous AURKA in KG1A and Kasumi‐1 cells following 12 h treatment with the indicated concentration of PROTACs. C) Relative AURKA protein levels in panel B were quantified using the ImageJ software. D) KG1A and Kasumi‐1 cells were treated with PROTAC cocktail (4 × 10 −6 m ) for 12 h. The expression of AURKA and related proteins were detected. E) KG1A cells were arrested with nocodazole (0.1 µg mL −1 ) for 16 h followed by PROTAC cocktail (4 × 10 −6 m ) treatment for indicated times. The expression of AURKA and related proteins were detected. F) TMT‐based quantitative proteomics after treatment with dAurA383 and dAurA450 cocktail (1 × 10 −6 m ) or the DMSO Vehicle for 6 h in KG1A cells. The differentially expressed proteins are presented in the volcano plot. G) Degradation of endogenous AURKA in KG1A and Kasumi‐1 cells following 12 h treatment with PROTAC cocktail (4 × 10 −6 m ), NEDD8‐activating enzyme inhibitor MLN4924 (1 × 10 −6 m ) or proteasomal inhibitor Bortezomib (25 × 10 −9 m ).

Article Snippet: In brief, DMSO, AURKA PROTACs, and ATRA (A9120, Solarbio) treated leukemia cells from the in vitro and in vivo assays were collected, counted, and washed with PBS.

Techniques: Concentration Assay, Software, Expressing, Quantitative Proteomics

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: Development of further BET targeting PROTACs. Brd2/Brd3/Brd4 pan-selective degrader ARV-771 (54) (a), and Brd3/Brd4 selective degrader MZP-54 (55) (b).

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: First ERRα (50) and RIPK2 (51) targeting VHL-recruiting PROTACs.

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: E3 ligase targeting PROTACs. Homo-PROTAC CM11 (57) inducing self-degradation of VHL (a), and CRBN-VHL Hetero-PROTACs inducing the selective degradation of CRBN (b).

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: Bcl-xL targeting PROTACs DT2216 (63) (a) and 64 (b), and ternary cocrystal structure of 64 bound to Bcl-xL and VCB (PDB 6ZHC ) highlighting PROTAC–protein interactions (c) and de novo PPIs (d).

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: ERα targeting VHL-recruiting PROTACs derived from DNA-encoded library screening.

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques: Derivative Assay, Library Screening

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: Amide-to-ester conversion BET protein targeting PROTACs recruiting VHL leading to improved pharmacokinetics and cellular potency.

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques: Drug discovery

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: Trivalent PROTACs. Trivalent degrader SIM1 (101) targeting both BDs of Brd4 via two incorporated BET ligands (a), and trivalent degrader DP-V-4 (102) simultaneously targeting EGFR and PARP proteins (b).

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: Photocaged PROTACs. Photocleavable DEACM-caged ERRα degrader 104 (a), and photocleavable DMNB-caged BET degrader 105 (b).

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: Enzymatic activated PROTACs. Quinone-caged HaloPROTAC 107 activated by NOQ1-mediated reduction (a) and generation of ROS via NOQ1-mediated reduction of β-lapachone (110) activating ROS-responsive HaloPROTAC 108 and BET degrader 109 (b).

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Chemical Society Reviews

Article Title: Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

doi: 10.1039/d2cs00387b

Figure Lengend Snippet: Oligonucleotide-containing PROTACs. Schematic peptide-based RNA-PROTAC 118 (a), mode of action of TRAFTACs as TF degradation technology (b) and schematic DNA-nucleotide containing TF degrader 119 (c).

Article Snippet: Hypothesising that increasing binding valency of PROTACs might enhance degradation and target selectivity, the Ciulli laboratory at Dundee in collaboration with the Daniels team at Promega developed trivalent BET targeting degraders by fusing a bivalent BET PROTAC degrader with a bivalent BET inhibitor.

Techniques:

Journal: Cancers

Article Title: Targeting Protein Degradation Pathways in Tumors: Focusing on their Role in Hematological Malignancies

doi: 10.3390/cancers14153778

Figure Lengend Snippet: The currently studied PROTACs in clinical trials.

Article Snippet: Moreover, BET PROTACs induced apoptosis at nanomolar concentrations regardless of the MCL cell-line sensitivity to ibrutinib (Mino, Z138, MAVER-1, Granta-519).

Techniques: