Journal: bioRxiv

Article Title: The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy

doi: 10.1101/2022.08.20.504641

Figure Lengend Snippet: (A) Dose-response cytotoxicity was analyzed using a methylthiazoletetrazolium (MTT) assay in samples from acute myeloid leukemia (AML) or T- and B-acute lymphoblastic leukemia (T- or B-ALL) patients treated with vehicle or increasing concentrations of THZ-P1-2 (1.6, 3.2, 6.4, 12.5, 25, 50, and 100 μM) for 72 h. Values are expressed as the percentage of viable cells for each condition relative to vehicle-treated cells. The IC 50 values for each patient sample are described. (B) Apoptosis was detected by flow cytometry in gated human Sca1 - CD45 + CD34 + (or CD117 + cells for NPM1 -mutant AMLs) of acute myeloid leukemia (AML) samples in a co-culture system using a FITC-annexin V/DAPI staining method. Cells were treated with vehicle, cytarabine (AraC, 250 and 500 nM), venetoclax (VEN, 100 and 500 nM), and/or THZ-P1-2 (3.2 or 6.4 μM) for 72 h. Bar graphs represent the mean ± SD of all the independent patients screened, each point represents a patient. The p values are indicated in the graphs; * p < 0.05, ** p < 0.01, *** p < 0.0001; ANOVA and Bonferroni post-test. (C) Correlation analysis of THZ-P1-2 response with clinical, laboratory, molecular, and metabolic characteristics of the samples. Note that THZ-P1-2 responsiveness (area under curve, AUC) was clusterized with markers of mitochondrial metabolic markers (Levels of MMP in the blasts – Blast_TMRE; Basal OCR and Max OCR). Data were processed using the Morpheus platform ( https://software.broadinstitute.org/morpheus/ ) (D) Association of THZ-P1-2 sensitivity (area under the curve, AUC), mutations in FLT3, NPM1 , and RUNX1 , and European LeukemiaNet (ELN) risk stratification. (E) Evaluation of long-term proliferation in neonatal cord blood (CB) CD34 + and primary AML mononuclear cells using a co-culture system. Data are expressed as mean ± SD of at least three independent experiments. The time points and p values are indicated in the graphs; * p < 0.05, ** p < 0.01, *** p < 0.0001; ANOVA and Bonferroni post-test. (F) Neonatal cord blood (CB) CD34 + cells were plated in cytokine-supplemented methylcellulose in the presence of vehicle or THZ-P1-2 (3.2 or 6.4 μM). Colonies were counted after 8-14 days of culture and are represented as the percent of vehicle-treated controls. Bars indicate the mean ± SD of at least three assays. (G-H) Mitochondrial membrane potential was detected by flow cytometry in gated human CD45 + CD34 + (or CD117 + for NPM1 -mutant AMLs)/annexin V - from samples from acute myeloid leukemia (AML) in a co-culture system using the TMRE staining method. Cells were treated with vehicle, cytarabine (AraC, 250 nM), venetoclax (VEN, 100 or 500 nM), and/or THZ-P1-2 (3.2 [alone or in combination with VEN 100 nM] or 6.4 μM) for 72 h. Bar graphs represent the mean ± SD of at least three independent experiments, each point represents a patient. The p values are indicated in the graphs; * p < 0.05, ** p < 0.01, *** p < 0.0001; ANOVA and Bonferroni post-test. (I) Differently expressed proteins obtained from THZ-P1-2 (THZ)-sensitive (n = 3) and THZ-resistant (n = 3) AML patients were included in the heatmap (all false discovery rate (FDR) q-values (FDR q) < 0.25). (J) The bar graph represents the normalized enrichment scores (NES) for Hallmark, Reactome, and Kegg gene sets with FDR q < 0.05. (K) GSEA plots for enriched molecular signatures in THZ-P1-2 (THZ) resistant vs . sensitive AML patient’s proteome are also shown. NES and FDR q are indicated.

Article Snippet: Venetoclax (ABT-199) was obtained from TargetMol (Target Molecule Corp., Boston, MA, USA) and diluted to a 50 mM stock solution in DMSO.

Techniques: MTT Assay, Flow Cytometry, Mutagenesis, Co-Culture Assay, Staining, Software

Journal: Nature Communications

Article Title: Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-X L by engaging a single-residue discrepancy

doi: 10.1038/s41467-024-45848-1

Figure Lengend Snippet: a Conformational comparison of BCL-2 bound by different ligands. The overall structure of BCL-2 is shown as cartoon. The α3 and α4 helices (F112-R139) from superposed structures of BCL-2-cp1 complex, BCL-2-BAX complex (PDB ID: 2XA0 ), BCL-2-venatoclax complex (PDB ID: 6O0K ) and ligand-free NMR structure of BCL-2 (PDB ID: 1GJH ) are colored in magenta, green, yellow and gray, respectively. The spatial locations of these two helices are marked by dashed lines to indicate their conformational changes. b The conformation of BAX (green) in complex with BCL-2. The BAX residues L59, L63, I66 and L70 are shown as sticks binding into the P1, P2, P3 and P4 pockets (marked with black arrows), respectively. c The conformation of venetoclax (yellow) in complex with BCL-2. d The conformation of cp1 (magenta) in complex with BCL-2. e Conformational comparison of BCL-X L bound by different ligands. The overall structure of BCL-X L is shown as cartoon. The α3 and α4 helices (F105-R132) from superposed crystal structures of ligand-free BCL-X L (PDB ID: 1AF3 ), BCL-X L -cp1 complex, BCL-X L -ABT-737 complex (PDB ID: 2YXJ ) and BCL-X L -BID complex (PDB ID: 4QVE ), are colored in gray, magenta, light blue and cyan, respectively. f The conformation of BID (cyan) in complex with BCL-X L. The BID residues I83, I86, L90 and V93 are shown as sticks binding into P1, P2, P3 and P4 pockets, respectively. g The conformation of ABT-737 (light blue) in complex with BCL-X L . h The conformation of cp1 (magenta) in complex with BCL-X L .

Article Snippet: The AbbVie team exploited co-crystal structures of BCL-2 and small-molecule inhibitors to guide their rational design of venetoclax (ABT-199), a first-in-class, highly specific inhibitor of BCL-2, and one of the first approved small-molecule tumor therapeutics that directly blocks a PPI – .

Techniques: Comparison, Binding Assay

Journal: Nature Communications

Article Title: Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-X L by engaging a single-residue discrepancy

doi: 10.1038/s41467-024-45848-1

Figure Lengend Snippet: a Locations of key residues in the BH3-binding groove of BCL-2. The four binding pockets (P1-P4) are marked by red ellipses. b Comparison of cp1-bound BCL-2 WT and BCL-2 G101V complexes. The overall structures of BCL-2 WT and G101V are shown as cartoon in limon and green, with the cp1 in complex with WT and G101V respectively colored in magenta and brown. c The conformation comparison of key BCL-2 residues G/V101, D103, F104, F112 and E152 in complexes with different ligands. The residues from BCL-2 WT-cp1, BCL-2 G101V-cp1, BCL-2 G101V-S55746 and BCL-2 G101V-venetoclax complexes are shown as sticks in gray, green, cyan and yellow, respectively. Conformation of key BCL-2 residues potentially involved in ligand-binding in the BCL-2 WT-cp1 complex (gray) as compared with those in the complexes of BCL-2 G101V-cp1 ( d , green), BCL-2 G101V-S55746 ( e , cyan) and BCL-2 G101V-venetoclax ( f , yellow). g Comparison of the binding positions of cp1, venetoclax and S55746 upon BCL-2. The MD simulation results showing chi1 angle distributions of BCL-2 residues F104 (left) and F112 (right) from the WT or G101V mutant in the states of ligand-free ( h , blue), cp1-bound ( i green), venetoclax-bound ( j red) and S55746-bound ( k magenta), respectively. Solid lines represent simulation (Sim) distribution of chi1 angles, while dotted lines indicate their experimental (Exp) status in the crystal structures. l Correlation between conformational changes of different BCL-2 residues. For residue 101, the amino acid type (G/V) was used as the state in simulation, with 0 for G and 1 for V. For D103, the chi2 angle was used as its state. While for the other residues, the chi1 angle was chosen as the state indicator. The color from red to blue indicates the correlation from high to low, while thickness of the orange arrows represents the strength of their correlation.

Article Snippet: The AbbVie team exploited co-crystal structures of BCL-2 and small-molecule inhibitors to guide their rational design of venetoclax (ABT-199), a first-in-class, highly specific inhibitor of BCL-2, and one of the first approved small-molecule tumor therapeutics that directly blocks a PPI – .

Techniques: Binding Assay, Comparison, Ligand Binding Assay, Mutagenesis, Residue

Journal: Nature Communications

Article Title: Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-X L by engaging a single-residue discrepancy

doi: 10.1038/s41467-024-45848-1

Figure Lengend Snippet: a Effects of different CPs on the viability of Jurkat cells with venetoclax as a positive control. b IC50 values of venetoclax, ABT-263, S55746 and TAT-conjugated CPs each against Jurkat, Daudi, El4 and RS4:11 cells, as measured after 48 h treatment by the CellTiter-Glo assay and flow cytometry. “NO” means no inhibition detected. IC50 values of venetoclax ( c ), S55746 ( d ) and cp3-TAT ( e ) against normal A549 cells (control) and those stably overexpressing BCL-2 WT or G101V mutant. Data are presented as mean ± S.D.; n = 6 biological replicates. f BAX inhibition assay of Jurkat cells treated by BIP-V5 at 50 μM for 24 h. The experiment was repeated 3 times independently with similar results. g Cytochrome C release assay of Jurkat cells treated with venetoclax (2.5 μM), cp4-TAT (20 μM), cp5-TAT (15 μM) for 12 h in the absence or presence of BIP-V5. The experiment was repeated 3 times independently with similar results.

Article Snippet: The AbbVie team exploited co-crystal structures of BCL-2 and small-molecule inhibitors to guide their rational design of venetoclax (ABT-199), a first-in-class, highly specific inhibitor of BCL-2, and one of the first approved small-molecule tumor therapeutics that directly blocks a PPI – .

Techniques: Positive Control, Glo Assay, Flow Cytometry, Inhibition, Control, Stable Transfection, Mutagenesis, Release Assay

Journal: Nature Communications

Article Title: Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-X L by engaging a single-residue discrepancy

doi: 10.1038/s41467-024-45848-1

Figure Lengend Snippet: a Representative results from the apoptosis assay of Jurkat cells using Annexin V-FITC/PI staining. Q4 region contains Annexin V − /PI − cells (live cells); Q3 shows Annexin V + /PI − cells (early apoptotic or apoptotic cells); Q2 represents Annexin V + /PI + cells (late apoptotic cells); Q1 contains Annexin V − /PI + cells (mechanical damaged cells). b Quantitative analysis of Jurkat cell apoptosis after 24 h treatment with different inhibitors. c The mitochondrial membrane potential (mtΔΨ) analysis of Jurkat cells after 4 h treatment with different inhibitors. d Statistical analysis of mtΔΨ (Q1/Q3). Data are presented as mean ± s.d.; n = 3 biological replicates; one-way ANOVA with Dunnett’s multiple comparisons test. The calculated P values were P = 0.053, P < 0.0001, P < 0.0001, P < 0.001 and P < 0.0001 for TAT, venetoclax, cp1-TAT, cp2-TAT and cp3-TAT, respectively. NS no significant difference. e A diagram showing the special binding mode of CPs and how CPs might overcome drug-resistant mutations.

Article Snippet: The AbbVie team exploited co-crystal structures of BCL-2 and small-molecule inhibitors to guide their rational design of venetoclax (ABT-199), a first-in-class, highly specific inhibitor of BCL-2, and one of the first approved small-molecule tumor therapeutics that directly blocks a PPI – .

Techniques: Apoptosis Assay, Staining, Membrane, Binding Assay