Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a Cytoplasmic HuR expression in breast cancer tissues with low or high grade. Patients with high-grade tumors (II-III or III, n = 42) have higher positively stained cytoplasmic HuR than those with low-grade tumors (II, n = 93) (* P = 0.0176, t -test). b Kaplan–Meier analysis of the distant disease-free survival of 134 patients comparing high and low cytoplasmic HuR. Patients with high cytoplasmic HuR have lower distant disease-free survival rate compared to those with low cytoplasmic HuR (** P = 0.0035, log-rank test). c , d Protein expression levels of HuR and downstream targets in MDA-MB-231 cells, cells with control sgRNA (sgControl) and two HuR KO clones. c Representative WB results from one experiment. d Quantified relative expression of HuR downstream target Bcl-2, Msi2, and XIAP. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA). e Growth curves of MDA-MB-231 cells, sgControl and two HuR KO clones. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA). f , g Colony formation of MDA-MB-231 cells, sgControl and two HuR KO clones. f colony numbers per well (*** P < 0.001, one-way ANOVA, n = 3). g Representative images of colonies. h , i Invasion assay in parental MDA-MB-231 cells, sgControl and two HuR KO clones. h Representative images of stained invaded cells, scale bars: 200 μm. i The number of invaded cells per image (*** P < 0.001, one-way ANOVA, n = 6). j , k Tumor initiation ( j ) and tumor growth ( k ) of MDA-MB-231 cells, sgControl and HuR KO1 clone in athymic nude mice. HuR KO1 is unable to engraft tumor in vivo ( n = 10, *** P < 0.001, log-rank test for tumor initiation and two-way ANOVA for tumor growth).

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: Expressing, Staining, Clone Assay, Invasion Assay, In Vivo

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a HTS in ∼2000 compounds from the NCI libraries and in-house compounds using FP assay. Shown here is the scattergram of compound activity expressed as % of inhibition. Median + 3 SD was used as a threshold to pick initial hits. KH-3 and Aza-9 are two top hits. KH-3B has no activity in the screening. b Chemical structures of KH-3 and KH-3B. c Dose–response curve of KH-3 and negative control KH-3B disrupting HuR–ARE Msi1 binding in FP assay using 10 nM HuR protein and 2 nM fluorescein-labeled Msi1 RNA ( n = 3). d Dose–response curve of KH-3 and KH-3B disrupting HuR–ARE Msi1 binding in ALPHA assay using 100 nM HuR RRM1/2 protein and 25 nM biotin-labeled Msi1 RNA ( n = 3). e SPR analysis of KH-3 binding to immobilized full-length HuR protein. Six doses were used and duplicated. f Computational docking of KH-3 and KH-3B to HuR RRM1/2. The protein is shown in surface representation with positive charges in blue and negative charges in red. KH-3 (green) and KH-3B (magenta) are shown in sticks. g Cellular thermal shift curves of HuR in MDA-MB-231 cells treated with DMSO or 20 μM KH-3, values are mean from two independent experiments. h Representative western blot results from one experiment. i , j Pull-down analysis of KH-3 disrupting ARE Msi1 oligo binding to endogenous HuR in MDA-MB-231 cells. Random oligo is used as a negative control of the assay and unlabeled ARE Msi1 oligo is used as a positive control. i Representative western blot result from one experiment. j Quantified relative HuR expression. Values are mean ± SD from n = 3 independent experiments (** P < 0.01, one-way ANOVA). k RNP IP analysis of HuR bound mRNAs affected by KH-3 in MDA-MB-231 cells. Isotype IgG is used as a negative control of HuR antibody. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA).

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: FP Assay, Activity Assay, Inhibition, Negative Control, Binding Assay, Labeling, Western Blot, Positive Control, Expressing

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a , b MTT-based cytotoxicity of KH-3 against a panel of TNBC cell lines ( a ) and MDA-MB-231 cells, sgControl and two HuR KO clones ( b ). c – h Half-life of Bcl-2, Msi2 and XIAP mRNA in MDA-MB-231 ( c – e ) and SUM159 ( f – h ) cells treated with 5 μg/mL actinomycin D together with DMSO, KH-3 or KH-3B. Data are mean ± SD from n = 3 independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: Clone Assay

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a – d Protein levels of Bcl-2, Msi2, XIAP, and HuR in MDA-MB-231 ( a , b ) and SUM159 ( c , d ) cells treated with DMSO, KH-3 or KH-3B at the indicated doses for 48 h. α-Tubulin is used as loading control. Representative western blot results from one experiment in MDA-MB-231 ( a ) and SUM159 ( c ) cells. Quantified relative expression of HuR and downstream target Bcl-2, Msi2 and XIAP in MDA-MB-231 ( b ) and SUM159 ( d ) cells. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: Western Blot, Expressing

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a – c Scratch assay in MDA-MB-231 and SUM159 cells treated with DMSO or KH-3. a , b Representative images of cell migration at 0 and 24 h after scratching with indicated treatment in MDA-MB-231 ( a ) and SUM159 ( b ) cells, scale bars: 50 μm. c Wound widths in two cell lines 24 h after scratching and treatment (*** P < 0.001, t -test, n = 3). d – f Invasion assay in MDA-MB-231 and SUM159 cells treated by DMSO, KH-3B or KH-3. d , e Representative images of stained invaded cells with indicated treatment in MDA-MB-231 ( d ) and SUM159 ( e ) cells, scale bars: 200 μm. f Invaded cell numbers per image in both cell lines with indicated treatment (*** P < 0.001, one-way ANOVA, n = 6). g The heatmap view of PCR pathway array focusing on invasion and metastasis related genes. The relative mRNA levels were presented as z score, each treatment was triplicated. h CDH1 luciferase reporter assay in HEK 293FT cells treated by DMSO, KH-3B or KH-3. Values are mean ± SD from n = 4 independent experiments (** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: Wound Healing Assay, Migration, Invasion Assay, Staining, Luciferase, Reporter Assay

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a Venn diagram depicting the number of targets identified in two independent RNA-seq experiments. FOXQ1 is a direct HuR target, which is also one of the top mRNAs decreased by KH-3 treatment. b Protein expression levels of HuR, FOXQ1 and E-cadherin in HMEC and a panel of TNBC cell lines. c , d Pull-down analysis of KH-3 disrupting ARE FOXQ1 oligo binding to endogenous HuR in MDA-MB-231 and SUM159 cells. c Representative western blot result from one experiment. d Quantified relative HuR expression. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, *** P < 0.001, one-way ANOVA). e , f RNP IP analysis of HuR bound FOXQ1 mRNA affected by KH-3 in MDA-MB-231 ( e ) and SUM159 ( f ) cells. Values are mean ± SD from three independent experiments (** P < 0.01, *** P < 0.001, one-way ANOVA). g , h Relative FOXQ1 mRNA levels in MDA-MB-231 ( g ) and SUM159 ( h ) cells treated with DMSO, KH-3 or KH-3B at the indicated time points. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA). i , j FOXQ1 3′-UTR luciferase reporter assay in MDA-MB-231 ( i ) and SUM159 ( j ) cells treated by DMSO, KH-3B or KH-3. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA).

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: RNA Sequencing Assay, Expressing, Binding Assay, Western Blot, Luciferase, Reporter Assay

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a , b Invasion assay in parental MDA-MB-231 cells, sgControl and two HuR KO clones transfected with control vector or vector containing FOXQ1 cDNA. a Representative images of stained invaded cells, scale bars: 200 μm. b The number of invaded cells per image (*** P < 0.001, one-way ANOVA, n = 6). c mRNA expression levels of FOXQ1, CDH1 and CD82 in parental MDA-MB-231 cells, sgControl and two HuR KO clones transfected with control vector or vector containing FOXQ1 cDNA. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, *** P < 0.001, two-way ANOVA). d , e Invasion assay in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with DMSO or 10 μM KH-3 treatment. d Representative images of stained invaded cells, scale bars: 200 μm. e The number of invaded cells per image (*** P < 0.001, one-way ANOVA, n = 6). f mRNA expression levels of FOXQ1, CDH1 and CD82 in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with treatment of DMSO or 10 μM KH-3. Values are mean ± SD from n = 3 independent experiments (** P < 0.01, *** P < 0.001, two-way ANOVA). g , h Protein expression levels of FOXQ1, Bcl-2, Msi2, β-catenin, and HuR in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with treatment of DMSO or KH-3 at the indicated doses for 48 h. α-Tubulin is used as loading control. g Representative western blot results from one experiment. h Quantified relative expression of HuR and downstream targets. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: Invasion Assay, Clone Assay, Transfection, Plasmid Preparation, Staining, Expressing, Western Blot

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a In vivo anti-tumor efficacy of KH-3 in MDA-MB-231 orthotopic xenograft model. KH-3 treatment significantly inhibits the tumor growth compared to the vehicle control ( n = 12, ** P < 0.01, *** P < 0.001, two-way ANOVA). b Protein expression levels of HuR and downstream targets in tumor tissues after treatment. α-Tubulin is used as loading control. c , d Kaplan–Meier analysis of the initiation of pulmonary metastases ( c ) and overall survival of mice ( d ) comparing treatment with vehicle control or KH-3 in an experimental metastasis model ( n = 9, * P < 0.05, ** P < 0.01, log-rank test). e Schematic of screening for HuR inhibitors that bind to HuR and block HuR function. f Proposed working model of KH-3 inhibiting breast cancer invasion and metastasis.

Article Snippet: Human TNBC cell line BT-549, Hs 578T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 as well as mouse TNBC cell line 4T1 were purchased from American Type Culture Collection (ATCC) and the 2LMP subclone which was generated from MDA-MB-231 formed lung metastasis in mice was a kind gift from Dr. Marc Lippman.

Techniques: In Vivo, Expressing, Blocking Assay

Journal: Journal of medicinal chemistry

Article Title: Simple Structural Modifications Converting a Bona Fide MDM2 PROTAC Degrader into a Molecular Glue Molecule: A Cautionary Tale in the Design of PROTAC Degraders

doi: 10.1021/acs.jmedchem.9b00846

Figure Lengend Snippet: (A). Structures of MDM2 inhibitors MI-1061 and MI-2103, the bona fide MDM2 degrader MD-222 and a putative MDM2 degrader MG-277. (B-D).Binding affinities of MI-1061 (B), MI-2103 (C) and MG-277 (D) to MDM2 by an optimized fluorescence-polarization (FP) competitive binding assay.

Article Snippet: All primers used for qRT-PCR were purchase from ThermoFisher and as follows: MDM2 (Hs01066930_m1; ThermoFisher); p21 (Hs00355782_m1; ThermoFisher); Puma (Hs00248075_m1; ThermoFisher); Bax (Hs00180269_m1; ThermoFisher), GSPT1 (Hs01093019_m1; ThermoFisher) and GAPDH (Hs02758991_g1; ThermoFisher).

Techniques: Binding Assay, Fluorescence, Competitive Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Simple Structural Modifications Converting a Bona Fide MDM2 PROTAC Degrader into a Molecular Glue Molecule: A Cautionary Tale in the Design of PROTAC Degraders

doi: 10.1021/acs.jmedchem.9b00846

Figure Lengend Snippet: (A-B) Western blotting analysis of MDM2 and p53 protein levels in RS4;11 cells after treatment with MI-2103, MG-277 and MD-222 for 1 h (A) or 2 h (B), and in p53 mutant RS4;11/IRMI-2 cell line after 2 h treatment (C). The cells were treated for 1 h or 2 h with the compounds at the indicated concentrations, and proteins were probed by specific antibodies. (D-G) Changes of mRNA levels of p53 downstream target genes, including MDM2, CDKN1A, PUMA and BAX were analyzed after treatment with 3 nM MG-277 or 10 nM MD-222 in the RS4;11 cell line by qRT-PCR.

Article Snippet: All primers used for qRT-PCR were purchase from ThermoFisher and as follows: MDM2 (Hs01066930_m1; ThermoFisher); p21 (Hs00355782_m1; ThermoFisher); Puma (Hs00248075_m1; ThermoFisher); Bax (Hs00180269_m1; ThermoFisher), GSPT1 (Hs01093019_m1; ThermoFisher) and GAPDH (Hs02758991_g1; ThermoFisher).

Techniques: Western Blot, Mutagenesis, Quantitative RT-PCR

Journal: Journal of medicinal chemistry

Article Title: Simple Structural Modifications Converting a Bona Fide MDM2 PROTAC Degrader into a Molecular Glue Molecule: A Cautionary Tale in the Design of PROTAC Degraders

doi: 10.1021/acs.jmedchem.9b00846

Figure Lengend Snippet: (A-D) Effect of knockdown of MDM2 on the cell growth inhibitory activity of MI-2103 and MG-277 in MDA-MB-231 and MDA-MB-468 cells after transfection with MDM2 siRNAs (C-D) or non-targeting siRNA control vector (A-B) for 2 days. (E) MDM2 knockdown efficiency was determined by Western blotting analysis.

Article Snippet: All primers used for qRT-PCR were purchase from ThermoFisher and as follows: MDM2 (Hs01066930_m1; ThermoFisher); p21 (Hs00355782_m1; ThermoFisher); Puma (Hs00248075_m1; ThermoFisher); Bax (Hs00180269_m1; ThermoFisher), GSPT1 (Hs01093019_m1; ThermoFisher) and GAPDH (Hs02758991_g1; ThermoFisher).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Journal of medicinal chemistry

Article Title: Simple Structural Modifications Converting a Bona Fide MDM2 PROTAC Degrader into a Molecular Glue Molecule: A Cautionary Tale in the Design of PROTAC Degraders

doi: 10.1021/acs.jmedchem.9b00846

Figure Lengend Snippet: (A-C) Structures of MC-215, MC-216 and MC-217, three control analogues of MG-277 formed by removal of fluorine and/or one or two chlorine atoms from the MI-2103 portion of MG-277. (D-E) Cell growth inhibitory activity of MG-277, MC-215, MC-216 and MC-217 in RS4;11 (D) and RS4;11/IRMI-2 (E) cell lines. (F) Binding affinity of the three analogues of MG-277 with MDM2 protein was determined by an FP assay.

Article Snippet: All primers used for qRT-PCR were purchase from ThermoFisher and as follows: MDM2 (Hs01066930_m1; ThermoFisher); p21 (Hs00355782_m1; ThermoFisher); Puma (Hs00248075_m1; ThermoFisher); Bax (Hs00180269_m1; ThermoFisher), GSPT1 (Hs01093019_m1; ThermoFisher) and GAPDH (Hs02758991_g1; ThermoFisher).

Techniques: Activity Assay, Binding Assay, FP Assay

Journal: Journal of medicinal chemistry

Article Title: Simple Structural Modifications Converting a Bona Fide MDM2 PROTAC Degrader into a Molecular Glue Molecule: A Cautionary Tale in the Design of PROTAC Degraders

doi: 10.1021/acs.jmedchem.9b00846

Figure Lengend Snippet: (A-B) Western blotting for GSPT1 protein level after 2 h treatment with MI-2103, MG-277 or MD-222 in MDA-MB-231 (A) and MDA-MB-468 (B) cells with or without lenalidomide (30 μM) to block binding with cereblon. (C-D) MDA-MB-231 (C) and MDA-MB-468 (D) cells were transfected with CRBN siRNAs targeting two different sequences or non-targeting siRNA control. Protein levels of GSPT1 and cereblon were determined by Western blot analysis after treatment with 0.1 μM MG-277 for 2 h or 24 h in the transfected cells. (E) Western blot analysis for GSPT1 protein level after 2 h treatment with MG-277 in the presence or absence of 10 μM MI-2103 (or DMSO as control) to block binding with MDM2 in MDA-MB-231 cells. (F) Western blotting for MDM2 and GSPT1 protein levels after 2 h treatment with MG-277 and three analogues (MC-215, MC-216 and MC-217) with weaker MDM2 binding affinity in RS4;11 cells. (G) RS4;11 cells were pre-incubated with a proteasome inhibitor, PR-171 or a Nedd8 inhibitor, MLN4924, or DMSO as control for 4 h to block proteasome or CRL function. GSPT1 protein level was measured by Western blotting after 2 h treatment with MI-2103, MG-277 or MD-222.

Article Snippet: All primers used for qRT-PCR were purchase from ThermoFisher and as follows: MDM2 (Hs01066930_m1; ThermoFisher); p21 (Hs00355782_m1; ThermoFisher); Puma (Hs00248075_m1; ThermoFisher); Bax (Hs00180269_m1; ThermoFisher), GSPT1 (Hs01093019_m1; ThermoFisher) and GAPDH (Hs02758991_g1; ThermoFisher).

Techniques: Western Blot, Blocking Assay, Binding Assay, Transfection, Incubation

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) uPAR is shown in a surface representation with residues colored based on hydrophobicity. More hydrophobic residues are colored brown while more hydrophilic residues are colored green. uPA is colored cyan and shown in cartoon. The sidechain of the four hotspots of uPA used in the pharmacophore analysis are shown in stick. (B) Experimental alanine scan of the uPAR•uPA binding pocket. The change in free energy between the mutated and wild-type complexes (ΔΔG) after mutation of the residue to alanine is color-coded. (C) Per-residue decomposition energies of the uPAR•uPA binding pocket. The total enthalpic contribution of each residue is color-coded. (D) Features of the pharmacophore model used to identify compounds that overlap with and mimic the hot spot residues of uPA. uPAR is shown in the background colored in white and shown in cartoon. uPA is shown in transparent cyan cartoon, with the five hot spot residues shown in stick. A pharmacophore model was used to assign features to four of the five hot spot residues (Ile-28 was excluded). The amine on the side chain of Lys-23 was assigned a positive ionizable feature (transparent red circle), while the aromatic rings of Tyr-24, Phe-25, and Trp-30 were assigned aromatic ring features (transparent yellow circles). Two separate pharmacophore features were assigned to each of the two rings on the indole on Trp-30.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Mutagenesis, Residue

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: A fingerprint method is used to compare how effectively compounds mimic receptor hot spots on uPAR. For each docked compound, MM-GBSA is used to calculate the per-residue decomposition energies between the compound and uPAR. The per-residue decompositions used are used to generate a fingerprint, where each position on the fingerprint corresponds to the interaction energy between uPAR and the compound of interest. This fingerprint is compared to two separate fingerprints of the native ligand uPA. The first uPA fingerprint is from an experimentally-determined alanine scanning of uPAR. The second is from the per-residue decomposition of the uPAR•uPA complex. Compounds are rank-ordered based on their Tanimoto distance with the fingerprints of uPA and ΔEGBTOT.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Residue

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) Residues used in the uPAR fingerprints are colored on the surface of uPAR as follows: (i) Experimental alanine scan (orange), (ii) decomposition (pink), (iii) both (green). uPA is transparently overlaid in cartoon, with the sidechain of hot spot residues in stick. (B) Among the top-ranking 500 compounds from each of the fingerprints generated from decomposition energies or experimental alanine scanning, the proportion of compounds that overlap with each fingerprint residue. (C) Single-concentration FP screen of compounds resulting from the virtual screen based on uPAR hot spots. Each compound was screened in duplicate at 50 μM concentration. Hit compound 1 (IPR-2797) is highlighted in green. (D) The binding mode of 1 in the uPAR•uPA binding pocket. The compound is shown in yellow. uPAR is shown in white cartoon, with the sidechain of hot spot residues shown in pink stick. uPA is shown in partial transparent cyan cartoon. The sidechain of the four hotspots on uPA are shown in stick and colored cyan. (E) Concentration-dependent FP assay measuring the inhibition of uPAR•AE147-FAM peptide interaction by 1.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Generated, Residue, Concentration Assay, Binding Assay, FP Assay, Inhibition

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) The virtual screening binding mode of 8 in the uPAR•uPA binding pocket. The compound is shown in yellow. uPAR is shown in white cartoon, with the sidechain of hot spot residues shown in pink stick. uPA is shown in partial transparent cyan cartoon. The sidechain of the four hotspots on uPA are shown in stick and colored cyan. (B) Derivatives of 8 were screened at a single 50 μM concentration via FP assay in duplicates. Further pursued hits are highlighted in green. (C) Chemical structures of the pursued derivative hits. (D) Concentration-dependent FP assay measuring the inhibition of uPAR•AE147-FAM peptide interaction by the derivative compounds. Each concentration point is measured in duplicates. At high concentrations, the compounds were insoluble and as such the data points were omitted from curve-fitting. (E) Concentration-dependent ELISA assay measuring inhibition of uPAR•uPAATF interaction by the derivative compounds. Each concentration point is measured in duplicates. At high concentrations, the compounds were insoluble and as such the data points were omitted from curve-fitting.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Concentration Assay, FP Assay, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) The virtual screening binding mode of 9 (IPR-2532) in the uPAR•uPA binding pocket. The compound is shown in yellow. uPAR is shown in white cartoon, with the sidechain of hot spot residues shown in pink stick. uPA is shown in partial transparent cyan cartoon. The sidechain of the four hotspots on uPA are shown in stick and colored cyan. (B) Derivatives of 9 were screened at a single 50 μM concentration via FP assay in duplicates. Further pursued hits are highlighted in green. (C) Chemical structures of the pursued derivative hits. (D) Concentration-dependent FP assay measuring the inhibition of uPAR•AE147-FAM peptide interaction by the derivative compounds. Each concentration point is measured in duplicates. At high concentrations, the compounds were insoluble and as such the data points were omitted from curve-fitting. (E) Concentration-dependent ELISA assay measuring inhibition of uPAR•uPAATF interaction by the derivative compounds. Each concentration point is measured in duplicates.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Concentration Assay, FP Assay, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) Single-concentration FP screen of compounds resulting from the virtual screen based on uPA hot spots. Each compound was screened in duplicate at 50 μM concentration. Hits that are pursued are highlighted in green while those with problematic moieties are highlighted in red. Chemical structures of the highlighted molecules are shown above. (B) Overlap between the predicted binding mode of the hit molecules and the uPA hotspots are highlighted. FP and microtiter ELISA assays were used to measure the Ki and IC50 of the compounds in inhibiting uPAR•AE147-FAM peptide and uPAR•uPAATF interactions, respectively. Serial dilution points were measured in duplicates. (C) MSTI-based thiol reactivity assay was performed in triplicates at 100 μM compound and 30 μM MSTI concentrations. (D) Compound stability of 26, 28, and 29 were tested in methanol, PBS, and in the presence of uPAR by HPLC-MS.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Serial Dilution

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) The binding mode of 26 (IPR-2992) in the uPAR•uPA binding pocket. The compound is shown in yellow. uPAR is shown in white cartoon, with the sidechain of hot spot residues shown in pink stick. uPA is shown in partial transparent cyan cartoon. The sidechain of the four hotspots on uPA are shown in stick and colored cyan. (B) The core of 26 was used to identify analogs at 5 positions. Among the analogs discovered was 30 (IPR-3011). (C) Derivatives of 26 were screened at a single 50 μM concentration via the uPAR•AE147-FAM peptide FP assay in duplicates. The parent compound 26 is highlighted in orange, while compound 30 is highlighted in green. (D) The binding mode of 30 (green) is overlaid on the binding mode of 26. The additional ring at R1 allows 30 to bind deeper in the uPAR•uPA pocket. (E) Concentration-dependent FP assay measuring the inhibition of uPAR•AE147-FAM peptide interaction by 26 and 30. Each concentration point is measured in duplicates. At high concentrations, 30 was insoluble and as such the data points were omitted from curve-fitting. (F) Concentration-dependent ELISA assay measuring inhibition of uPAR•uPAATF interaction by 26 and 30. Each concentration point is measured in duplicates. At high concentrations, 30 was insoluble and as such the data points were omitted from curve-fitting. (G) MST experiment was performed with 40 nM NT-495-labeled uPAR and multiple concentrations of 26. MST concentration-response curve of the interaction between uPAR and 26 are shown. (H) MST experiment was performed with 40 nM NT-495-labeled uPAR and multiple concentrations of 30. MST concentration-response curve of the interaction between uPAR and 30 are shown. At high concentrations, 30 is insoluble and the high concentration points, in light orange, are omitted from curve fitting.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Concentration Assay, FP Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) The virtual screening binding mode of 28 in the uPAR•uPA binding pocket. The compound is shown in yellow. uPAR is shown in white cartoon, with the sidechain of hot spot residues shown in pink stick. uPA is shown in partial transparent cyan cartoon. The sidechain of the four hotspots on uPA are shown in stick and colored cyan. (B) Derivatives of 28 were screened at a single 50 μM concentration via FP assay in duplicates. Further pursued hits are highlighted in green. (C) Chemical structures of the pursued derivative hits. (D) Concentration-dependent FP assay measuring the inhibition of uPAR•AE147-FAM peptide interaction by the derivative compounds. Each concentration point is measured in duplicates. At high concentrations, the compounds were insoluble and as such the data points were omitted from curve-fitting.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Concentration Assay, FP Assay, Inhibition

Journal: Journal of chemical information and modeling

Article Title: Mimicking Native Interactions for Small-Molecule Inhibitors of Tight Protein-Protein Interactions

doi: 10.1021/acs.jcim.7b00181

Figure Lengend Snippet: (A) The virtual screening binding mode of 29 in the uPAR•uPA binding pocket. The compound is shown in yellow. uPAR is shown in white cartoon, with the sidechain of hot spot residues shown in pink stick. uPA is shown in partial transparent cyan cartoon. The sidechain of the four hotspots on uPA are shown in stick and colored cyan. (B) Derivatives of 29 were screened at a single 50 μM concentration via FP assay in duplicates. Further pursued hits are highlighted in green. (C) Chemical structures of the pursued derivative hits. (D) Concentration-dependent FP assay measuring the inhibition of uPAR•AE147-FAM peptide interaction by the derivative compounds. Each concentration point is measured in duplicates. At high concentrations, the compounds were insoluble and as such the data points were omitted from curve-fitting. (E) Concentration-dependent ELISA assay measuring inhibition of uPAR•uPAATF interaction by the derivative compounds. Each concentration point is measured in duplicates. At high concentrations, the compounds were insoluble and as such the data points were omitted from curve-fitting. (F) MST experiment was performed with 40 nM NT-495-labeled uPAR and multiple concentrations of 29. MST concentration-response curve of the interaction between uPAR and 29 are shown. At high concentrations, 29 is insoluble and the high concentration points, in light blue, are omitted from curve fitting.

Article Snippet: Following incubation for 30 min and subsequent washing steps, biotinylated human uPAR antibody (1:3000 dilution of 0.2 mg·mL –1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% bovine serum albumin (BSA) was added to the wells (100 μL/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Concentration Assay, FP Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Labeling

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Chemical structures of the parental BET protein family inhibitor (JQ1) and its derivative (ZZ1) featuring an appended chemical tag conferring degrader activity. b) HiBiT-BRD4 assay results for Jurkat cells pre-treated with the indicated inhibitors for 1 h, followed by treatment with ZZ1 for 5 h. c) Ubiquitin-proteasome system (UPS)-focused CRISPR screen for BRD4 BD1 -eGFP stability in K562-Cas9 cells treated with 1 µM ZZ1 for 16 h. d) Cartoon of the UBE2H∼Ub-bound YPEL5-GID/CTLH E3 ligase catalytic assembly highlighting its functional modules. e) In vitro ubiquitylation assay of fluorescently labeled *BRD4 BD1 (asterisk denotes an N-terminal FAM label) determining the minimal catalytic assembly sufficient for ZZ1-dependent activity. The examined GID/CTLH E3 ligases comprised the catalytic core (catalytic and scaffolding modules) alone or assembled with substrate regulatory subunits WDR26 and YPEL5. Reactions were quenched after 45 min. See for details of the GID/CTLH E3 ligase architecture and assays with a full suite of GID/CTLH E3 assemblies. f) Native gel mobility shift assay probing ZZ1-induced *BRD4 BD1 engagement by the WDR26 dimer or WDR26-YPEL5. Transparent regions in the cartoon represent truncated WDR26 domains. The truncations prevent higher-order oligomerization).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Activity Assay, Ubiquitin Proteomics, CRISPR, Functional Assay, In Vitro, Ubiquitin Assay, Labeling, Scaffolding, Mobility Shift

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Western blots showing BRD4 and BRD3 degradation in MOLT-4 cells after 5 h treatment with ZZ1. b) Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 h treatment with 1 µM of ZZ1. c) Identifying of the BRD4 region required for ZZ1-induced degradation with a cellular fluorescent reporter assay. The examined reporters were either the isolated BRD4 bromodomains (BD1 or BD2) or a tandem construct comprising BD1 and BD2 connected by the intervening native sequence (BRD4 BD1+BD2 ).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Western Blot, Mass Spectrometry, Reporter Assay, Isolation, Construct, Sequencing

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Color-coded guide to the GID/CTLH E3 subunits and their reported functions. b) Schematic illustrating the architecture of the GID/CTLH E3 ligases, which share a common catalytic core that associates with divergent auxiliary subunits enabling substrate targeting. WDR26 acts as a supramolecular assembly factor by connecting two copies of the catalytic core (either alone or bound to GID4-ARMC8) into a singular giant oval structure with a large hollow center. Each WDR26 homodimer in the supramolecular assembly can bind a single copy of YPEL5, yielding the YPEL5-GID/CTLH E3. Subunits are colored according to the guide in (a). c) Identifying E3 ligase leveraged by ZZ1 with in vitro ubiquitylation assays. The suite of GID/CTLH E3 assemblies shown in (b) was tested for activity towards fluorescent BRD4 bromodomain substrates, either in isolation (BRD4 BD1 and BRD4 BD2 ) or in tandem (BRD4 BD1+BD2 ). Asterisk denotes the fluorescent FAM label appended to substrates’ N-termini. All reactions were quenched after 45 min. d) Western blots showing BRD4 degradation in WT or YPEL5-KO Jurkat cells treated with the indicated concentration of ZZ1 for 5 h. e) Western blots showing BRD4 degradation in WT or WDR26-KO HEK293T cells treated with the indicated concentration of ZZ1 for 5 h. f) Western blots showing BRD4 degradation in HEK293T (YPEL5 low ) or TC-71 (YPEL5 high ) cells treated with the 2 µM of ZZ1 for 5 h. g) Co-immunoprecipitation of FLAG-tagged BRD4 and YPEL5-WDR26-containing GID/CTLH E3 in the presence of ZZ1. FLAG-tagged BRD4 transfected cells were preincubated with the proteasomal pathway inhibitor (bortezomib) for 1 h to prevent BRD4 degradation. h) In vitro ubiquitylation assay as in (b) but performed with the endogenous NMNAT1 substrate to recapitulate its previously reported GID/CTLH E3-dependent regulation. In contrast to its essential role in ZZ1-induced BRD4 ubiquitylation, YPEL5 acts as an inhibitor of NMNAT1 targeting. Reactions were quenched after 45 min. i) Previous cryo-EM structures of NMNAT1- and YPEL5-bound GID/CTLH E3 assemblies (EMD-18175 and EMD-18170, respectively) fit with segmented focused-refined maps of relevant modules (EMD-18345 and EMD-18316, respectively) explaining the biochemically defined mode of NMNAT1 targeting: the hexameric NMNAT1 and YPEL5 are engaged by the overlapping binding sites on dimeric WDR26 modules. Consequently, YPEL5 sterically blocks WDR26-mediated NMNAT1 recruitment.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: In Vitro, Activity Assay, Isolation, Western Blot, Concentration Assay, Immunoprecipitation, Transfection, Ubiquitin Assay, Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Cryo-EM map of the neosubstrate recognition complex (YPEL5-GID/CTLH E3-ZZ1-BRD4 BD1 ) resolved to 12 Å and fit with prior structures (extracted from PDB: 7NSC , 8PJN , 8QBN , 3MXF ) and AlphaFold models of the constituent GID/CTLH modules. b) Map of the YPEL5-WDR26 module-ZZ1-BRD4 BD1 ternary complex resolved to 3.4 Å and sharpened with DeepEMhancer . Close-up highlights additional electron density at the interface of YPEL5 and BRD4 BD1 corresponding to the ZZ1 degrader. c) Atomic model of the ZZ1-induced ternary complex depicting the overall YPEL5-WDR26 receptor module architecture and its mode of BRD4 BD1 engagement. d) Close-up of YPEL5 in complex with ZZ1-BRD4 BD1 overlayed with lenalidomide-bound CRBN CTD (PDB: 4TZ4 ) illustrating the structural similarity of their ligand-binding domains. Both domains are stabilized by coordination of a zinc atom and contain a central groove that binds ligands. e) YPEL5 and CRBN (PDB: 5FQD ) MGD ternary complexes have divergent modes of degrader engagement and neosubstrate positioning.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep, Ligand Binding Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Flowchart of the cryo-EM data processing workflow generating the focused-refined map of the ternary complex comprising ZZ1-SO 2 H c-Glue, YPEL5-WDR26 E3 receptor module and BRD4 BD1 neosubstrate. The scale bar in the motion-corrected representative micrograph corresponds to 300 Å. b) The final post-processed map color-coded to illustrate variations in its local resolution. c) Gold-standard Fourier shell correlation (FSC) plot. The dotted line represents 0.143 cut-off criterion for estimating nominal resolution.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) In vitro ubiquitylation assays testing specificity of ZZ1-induced ubiquitylation towards bromodomains of BET protein family members that all bind the parental substrate-recruiting JQ1 handle of ZZ1. b) Superposition of the YPEL5 structure with that of the thalidomide binding domain of CRBN (CRBN CTD ) engaging an Immunomodulatory Drug (IMiD) MGD (PDB: 4TZ4). The close-up highlights CRBN residues forming the IMiD-engaging hydrophobic site (the “tri-Trp pocket”) and the corresponding YPEL5 residues (shown as sticks). Despite adopting a homologous fold, YPEL5 does not possess two out of three residues critical for IMiD binding. c) Examining ligand-binding preference of YPEL5 by performing in vitro BRD4 BD1 ubiquitylation assay with ZZ1 and CRBN-based PROTACs employing the BRD4 ligand JQ1. d) Analysis of BRD4 BD1 -eGFP degradation in K562 stability reporter cells treated with indicated compounds or compound combinations (co-treatment with equimolar mixtures).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: In Vitro, Binding Assay, Ligand Binding Assay, Ubiquitin Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Structure of the ternary complex illustrating electrostatically-driven interactions between the negatively charged sulfinic acid moiety of ZZ1-SO 2 H and the basic bottom of the YPEL5 binding groove (represented as an electrostatic potential surface). b) Intact mass spectrometry demonstrates conversion of the sulfonyl fluoride moiety of ZZ1 (middle) to sulfinic acid (right) after incubation in a DTT-containing buffer. The position of the transformed group is indicated in the degrader’s chemical structure (left). Peaks corresponding to lower molecular weight species correspond to ZZ1 and ZZ1-SO 2 H derivatives formed upon hydrolysis of their tert -butyl ester. c) Stimulation of YPEL5-GID/CTLH E3-dependent in vitro ubiquitylation of *BRD4 BD1 . ZZ1, its sulfinic acid derivative, (ZZ1-SO 2 H) and its sulfonic acid derivative (ZZ1-SO 3 H) were tested. d) FP assay quantifying the propensity of ZZ1-SO 2 H to induce the ternary complex formation. Binding of *BRD4 BD1 to the YPEL5-WDR26 module upon degrader titration results in a dose-dependent increase of fluorescence polarization. Fitting polarization values to the “[agonist] vs. response” model yielded the half-maximal effective concentration (EC 50 ). e) HPLC analysis of intracellular levels of ZZ1 and its acidic metabolites. Jurkat cells were treated with 5 µM ZZ1 for 5 h.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Binding Assay, Mass Spectrometry, Incubation, Transformation Assay, Molecular Weight, In Vitro, FP Assay, Titration, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Close-up of electron density (gray transparent) in the cryo-EM structure of the ternary complex (shown in ) corresponding to the ZZ1-SO 2 H chemical tag and the surrounding YPEL5 residues, along with their atomic coordinates (sticks). Absence of continuous density between the sulfinic acid and neither YPEL5 nucleophilic amino acid side chains suggests the non-covalent ZZ1 mode-of-action. b) Intact mass spectrometry analysis testing formation of potential ZZ1-induced covalent adducts between YPEL5 (within the YPEL5-WDR26 subcomplex) and BRD4 BD1 . c) Real-time FP assay probing kinetics of ternary complex formation in the DTT-containing buffer induced by ZZ1, ZZ1-SO 2 H, or ZZ1 pre-incubated in the FP buffer. Polarization signal was measured over time after combining the protein mix (*BRD4 BD1 and YPEL5-WDR26) prepared in the DTT-containing buffer with the degrader compounds. d) Schematic of the proposed mechanism of sulfonyl fluoride conversion to sulfinic acid triggered by nucleophilic thiol groups of reducing agents, such as DTT ( in vitro ) or GSH (in cells). e) Real-time FP assay testing effect of DTT on the rate of the ZZ1-SO 2 H-induced ternary complex formation. The varying DTT concentrations during the experiment were controlled by the composition of the buffer used for preparing the protein mix (*BRD4 BD1 and YPEL5-WDR26) prior to degrader addition. f) HiBiT-BRD4 assay results for Jurkat cells pre-treated with 100 µM GSH for 2 h, followed by treatment with the indicated compounds for 5 h.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep, Mass Spectrometry, FP Assay, Incubation, In Vitro

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Close-up of cryo-EM density corresponding to the YPEL5-engaging chemical tag of ZZ1-SO 2 H (compound coordinates shown as sticks). b) Molecular details of the ternary complex interface highlighting the constellation of YPEL5 residues engaging ZZ1-SO 2 H and those involved in direct interactions with BRD4 BD1 . The hydrogen bonds are depicted as gray dashes. c) In vitro ubiquitylation assay probing YPEL5 residues shown in (b) involved in: (1) anchoring the sulfinic acid moiety (T63, K95), (2) contacts with the degrader phenyl ring and BRD4 hydrophobic sidechains (L62), and (3) direct interactions with BRD4 (R41). Reactions were quenched after 30 minutes. SDS-PAGE gels were imaged by a fluorescence scan and stained with Coomassie to visually inspect YPEL5 levels (bottom). d) Cellular BRD4 degradation assay testing the structurally visualized binding mode. The impact of YPEL5 mutations on ZZ1 potency is illustrated as the ratio of DC 50 values between mutant and WT YPEL5-expressing Jurkat cells. Plots used for DC 50 measurements are presented in . e) Competitive FP assay probing cooperativity within the ZZ1-SO 2 H-induced ternary complex. BRD4 BD1 -bound fluorescent JQ1* tracer was displaced by titration of unlabeled competitors reducing fluorescence polarization. The extent of cooperativity was determined by calculating the ratio of IC 50 values (estimated by fitting polarization values to the “[inhibitor] vs. response” model) in the absence and presence of excess YPEL5-WDR26 (apparent cooperativity factor α app ).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep, In Vitro, Ubiquitin Assay, SDS Page, Fluorescence, Staining, Degradation Assay, Binding Assay, Mutagenesis, Expressing, FP Assay, Titration

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) HiBiT-BRD4 assay results for WT and mutant YPEL5-expressing Jurkat cells treated with the indicated compounds for 5 h. The plots comparing the estimated DC 50 values are presented in . b) FP assay for establishing the competitive ligand displacement experiment probing cooperative ternary complex formation . Polarization values upon BRD4 BD1 titration to the fluorescent JQ1 tracer (FAM-JQ1) were fit to the one-site binding model to estimate the BRD4 BD1 -JQ1 affinity (equilibrium dissociation constant, K D ). c) Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 h treatment with 1 µM ZZ2. d) Intact mass spectrometry demonstrates conversion of the sulfonyl fluoride moiety of ZZ2 (left) to sulfinic acid (right) after incubation in a DTT-containing buffer. Peaks corresponding to lower molecular weight species correspond to ZZ2 and ZZ2-SO 2 H derivatives formed upon hydrolysis of their tert -butyl ester. e) Close-up of ternary complex structures induced by ZZ1-SO 2 H and ZZ2-SO 2 H highlighting their common binding mode. YPEL5 and BRD4 residues involved in protein-protein and/or protein-degrader contacts are shown as sticks. Spheres represent water molecules, while dashes denote hydrogen bonds.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Mutagenesis, Expressing, FP Assay, Titration, Binding Assay, Mass Spectrometry, Incubation, Molecular Weight

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Electrostatic potential surface of the ZZ1-SO 2 H-bound YPEL5 groove showcasing a vacant basic pocket (outlined with a red dash) adjacent to the unsubstituted position of the degrader’s chemical tag (indicated by an arrow). b) Chemical structure of ZZ2 highlighting the incorporated chloro group (red) at the second ortho -position of ZZ1’s chemical tag. c) HiBiT-BRD4 assay results for Jurkat cells treated with the indicated compounds for 5 h. d) Qualitative comparison of ZZ1-SO 2 H and ZZ2-SO 2 H ability to trigger in vitro YPEL5-GID/CTLH E3-catalyzed *BRD4 BD1 ubiquitylation. ZZ2-SO 2 H was generated by pre-incubation of ZZ2 in the DTT-containing buffer . e) FP assay quantifying propensity of ZZ1-SO 2 H and ZZ2-SO 2 H to promote ternary complex formation. Note that polarization values obtained upon ZZ2-SO 2 H titration fit the “[agonist] vs. response” model but its superior MGD activity precludes accurate estimation of EC 50 . f) Real-time FP assay testing rates of ternary complex formation triggered by ZZ1 and ZZ2 as well as their acidic derivatives. Fluorescence polarization was monitored over time upon mixing *BRD4 BD1 and YPEL5-WDR26 with different versions of the degraders. g) Close-up of the ZZ2-SO 2 H-induced ternary complex cryo-EM structure, resolved to 3.4 Å and sharpened with DeepEMhancer. The images highlight the overall fit of the degrader’s chemical tag (left) and the filling of the vacant YPEL5 basic pocket by the introduced chloro group according to the structure-based design (right).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Comparison, In Vitro, Generated, Incubation, FP Assay, Titration, Activity Assay, Fluorescence, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Flowchart of the cryo-EM data processing workflow generating the focused-refined map of the ternary complex comprising the improved ZZ2-SO 2 H c-Glue, YPEL5-WDR26 E3 receptor module and BRD4 BD1 neosubstrate. The scale bar in the motion-corrected representative micrograph corresponds to 300 Å. b) The final post-processed map color-coded to illustrate variations in its local resolution. c) Gold-standard Fourier shell correlation (FSC) plot. The dotted line represents 0.143 cut-off criterion for estimating nominal resolution.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep

Journal: iScience

Article Title: Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia

doi: 10.1016/j.isci.2022.104170

Figure Lengend Snippet: Novel SHIP1 agonists and their relative activity on SHIP1 and SHIP2 (A) Structures of novel SHIP1 agonists. Percentage increase of phosphatase activity for (B) SHIP1 and (C) SHIP2 enzyme in the malachite green assay. The assay was performed at the indicated mM concentration with the indicated agonist or its vehicle control (0) with 100μM PI(3,4,5)P 3 -diC8. (Data are representative of 2 independent experiments. Bars indicate mean with ±SEM. The significance of agonism (or inhibition) for each compound vs. vehicle was assessed for all concentration tested via a two-way ANOVA. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗p < 0.05).

Article Snippet: Malachite Green Phosphatase Release Assays (Echelon Biosciences) were performed with recombinant human truncated SHIP1 (tSHIP1) ( ) and SHIP2 (Echelon Biosciences).

Techniques: Activity Assay, Malachite Green Assay, Concentration Assay, Control, Inhibition

Journal: iScience

Article Title: Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia

doi: 10.1016/j.isci.2022.104170

Figure Lengend Snippet: K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.

Article Snippet: Malachite Green Phosphatase Release Assays (Echelon Biosciences) were performed with recombinant human truncated SHIP1 (tSHIP1) ( ) and SHIP2 (Echelon Biosciences).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Fluorescence, Generated, FP Assay, Concentration Assay

Journal: iScience

Article Title: Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia

doi: 10.1016/j.isci.2022.104170

Figure Lengend Snippet: K306 agonism does not require the C2 domain to agonize SHIP1 (A) Structure of the tSHIP1, SHIP1-Enzyme (S1-Enz), SHIP1ΔC2 (S1ΔC2), and SHIP2-Enzyme (S2-Enz) constructs. (B) Malachite Green Phosphatase Release assay measurements of K306 or AQX-MN100 agonism on purified SHIP1ΔC2. Data in (B) are representative of three independent experiments. Bars indicate mean ±SEM. The significance of agonism for each compound vs. vehicle was assessed for all concentration tested via a two-way ANOVA∗∗∗∗p < 0.0001, ∗∗∗p < 0.001).

Article Snippet: Malachite Green Phosphatase Release Assays (Echelon Biosciences) were performed with recombinant human truncated SHIP1 (tSHIP1) ( ) and SHIP2 (Echelon Biosciences).

Techniques: Construct, Release Assay, Purification, Concentration Assay

Journal: iScience

Article Title: Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia

doi: 10.1016/j.isci.2022.104170

Figure Lengend Snippet:

Article Snippet: Malachite Green Phosphatase Release Assays (Echelon Biosciences) were performed with recombinant human truncated SHIP1 (tSHIP1) ( ) and SHIP2 (Echelon Biosciences).

Techniques: Virus, Recombinant, Plasmid Preparation, Activity Assay, Fluorescence, Protein Extraction, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Transfection, Negative Control, In Vitro, Generated, Stable Transfection, Expressing, Software, Imaging, Selection