Journal: Nature chemical biology

Article Title: Selective Inhibitors of mTORC1 Activate 4EBP1 and Suppress Tumor Growth

doi: 10.1038/s41589-021-00813-7

Figure Lengend Snippet: a) Representation of binding sites for FKBP12-rapamycin and active-site inhibitors in mTORC1 and mTORC2. Rapamycin has reduced affinity for mTORC2 due to partial occlusion of the FKBP12-rapamycin binding (FRB) domain, while active-site inhibitors have similar affinity for both complexes. b) Schematic representation of components of the bi-steric mTORC inhibitors: the rapamycin core (R), with sites of chemical modification shown as colored marks; the linker, with sites of attachment indicated by geometry of components and green and red color; and the active-site inhibitor analog (ASI). c) Structure of the rapamycin core, showing the linker attachment sites at C40 (green) and C26 (red), and the core modification sites at C32 (pink) and C16 (orange). d) Structures of the bi-steric molecules. The C40-linked bi-steric inhibitor RMC-4627 (BiS-13x) is shown in full, with modifications resulting in the inhibitors RMC-4287 (BiS-NS; replacement of the PP242 active-site inhibitor with MLN0128) and RMC-4745 (Bi-35x; methylcarbamate modification at C16) indicated. The C26-linked bi-steric inhibitor RMC-4529 (BiS-31x) is shown in full, with the C32 (R)-methoxy modification indicated in pink and the C26 oxime linker attachment indicated in red. e) Schematic representation of the design approach combining different linkers, active-site inhibitors, and core modifications to increase selectivity for mTORC1 over mTORC2 while maintaining potency. f) Levels of p4EBP1 T37/T46, pS6K T389, and pAKT S473 determined by MesoScale Discovery (MSD) or AlphaLISA platforms for whole cell lysates from MDA-MB-468 cells incubated with increasing concentrations of indicated compounds for two hours. These are graphs of representative experiments in which each data point is the mean of technical duplicates and normalized to vehicle control, with error bars representing SD. Independent replicate experiments: Rapamycin, n=5; MLN0128, n=7; RMC-4287, n=10; RMC-4627, n=11; RMC-4529, n=5; RMC-4745; n=5.

Article Snippet: mTOR substrate phosphorylation in MDA-MB-468 and MCF-7 cells was assayed using AlphaLISA SureFire Ultra kits for p-4EBP1 Thr37/46, p-P70S6K Thr389, and p-AKT1/2/3 Ser473 (PerkinElmer); and MesoScale Discovery Multi-Array Assay Systems for Phospho-4E-BP1 (Thr37/46) and Phospho-AKT (Ser473) (MSD).

Techniques: Binding Assay, Modification, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2

doi: 10.1016/j.jbc.2022.102803

Figure Lengend Snippet: The TSP1 domain of CCN5 is sufficient to inhibit phosphorylation of AKT and CCN2-induced cell migration. Panel ( A ) demonstrates concentration-effect curves of HHS-CCN5(III)- and HHS-CCN5(FL)-mediated inhibition of phospho-AKT (Ser473) levels in A549 human adenocarcinoma epithelial cells following 60 min incubation with the indicated proteins. The His-Halo-Sumo (HHS) fusion partner alone did not display any efficacy. Panel ( B ) demonstrates concentration-effect curves of Alb-CCN5(III)- and Alb-CCN3(III)-stimulated inhibition of phospho-AKT (Ser473) levels in A549 cells following 60 min incubation with the indicated proteins. The data in panels ( A and B ) represent the mean ± SEM (n = 3 independent experiments assayed in three replicates for each data point) using a TR-FRET–based or AlphaLISA-based immunoassay of cellular phospho-AKT (Ser473) contents. The data were subjected to curve fitting by four-variable nonlinear regression in GraphPad Prism. Panels ( C ) and ( D ) demonstrate concentration-effect curves of HHS-CCN5(III)- and Alb-CCN3(III)-mediated inhibition of CCN2 (5 μg/ml)-stimulated migration of Rat2 fibroblasts following 20 h incubation. The HHS fusion partner alone did not show any efficacy in the migration of Rat2 fibroblasts. Cell migration was determined using multiscreen migration assay (Boyden chamber principle). The data represent the mean ± SEM (n = 3 independent experiments assayed in four replicates for each data point). The data were subjected to curve fitting by four-variable nonlinear regression in GraphPad Prism. Alb, albumin; CCN, Cellular Communication Network; HHS, His-Halo-Sumo; TSP1, thrombospondin type 1 repeat.

Article Snippet: The cells were subsequently lysed in lysis buffer provided by the AlphaLISA SureFire Ultra phospho-AKT (Ser473) and phospho-ERK1/2 (Thr202/Tyr204 and Thr185/Tyr187) Assay Kits (PerkinElmer, Cat. No ALSU-PAKT-B-HV 100 test and ALSU-PERK-A-HV 100 test), transferred to a 96-well AlphaPlate (PerkinElmer), and analyzed with the sandwich bead–based AlphaLISA immunoassay (AlphaLISA Surefire Ultra immunoassay, PerkinElmer), according to the manufacturer’s instructions.

Techniques: Migration, Concentration Assay, Inhibition, Incubation

Journal:

Article Title: Cripto-1 Activates Nodal- and ALK4-Dependent and -Independent Signaling Pathways in Mammary Epithelial Cells

doi: 10.1128/MCB.22.8.2586-2597.2002

Figure Lengend Snippet: MAPK and AKT activation by CR-1ΔC in EpH-4 cells. Serum-starved EpH-4 cells were stimulated with recombinant CR-1ΔC (200 ng/ml) or EGF (50 ng/ml) protein and analyzed by Western blot (WB) analysis. MW, molecular weight in thousands; C, control.

Article Snippet: To ensure equal loading of proteins the blots were stripped and reprobed with Smad-2/Smad-3 (2 μg/ml) (Upstate Biotechnology), total MAPK (1:1,000) (Biolab Laboratories), or total AKT (1:1,000) (Biolab Laboratories) rabbit polyclonal antibodies.

Techniques: Activation Assay, Recombinant, Western Blot, Molecular Weight

Journal:

Article Title: Cripto-1 Activates Nodal- and ALK4-Dependent and -Independent Signaling Pathways in Mammary Epithelial Cells

doi: 10.1128/MCB.22.8.2586-2597.2002

Figure Lengend Snippet: MAPK and AKT phosphorylation by CR-1ΔC in MC3T3-E1 cells. (A) RT-PCR using specific primers for ALK4 (435 bp) in EpH-4 WT and MC3T3-E1 cells. A 100-bp DNA marker is shown in the first lane. (B and C) Serum-starved MC3T3-E1 cells were stimulated with CR-1ΔC (200 and 400 ng/ml) or EGF (50 ng/ml) recombinant protein and analyzed by Western blot (WB) analysis. MW, molecular weight in thousands; C, control.

Article Snippet: To ensure equal loading of proteins the blots were stripped and reprobed with Smad-2/Smad-3 (2 μg/ml) (Upstate Biotechnology), total MAPK (1:1,000) (Biolab Laboratories), or total AKT (1:1,000) (Biolab Laboratories) rabbit polyclonal antibodies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Recombinant, Western Blot, Molecular Weight

Journal:

Article Title: Cripto-1 Activates Nodal- and ALK4-Dependent and -Independent Signaling Pathways in Mammary Epithelial Cells

doi: 10.1128/MCB.22.8.2586-2597.2002

Figure Lengend Snippet: Smad-2, MAPK, and AKT phosphorylation by CR-1ΔC in EpH-4 WT and EpH-4 N cells transiently transfected with a dominant-negative ALK4-2-Flag receptor. EpH-4 WT and EpH-4 N cells transiently transfected with ALK4-2-Flag or pCI-neo plasmid were stimulated with CR-1ΔC, EGF, or Activin A recombinant protein and cell lysates were assessed by Western blot (WB) analysis. (A) EpH-4 N cells transfected with pCI-neo (4 μg) (lanes 1, 2, and 4) and ALK4-2 (4 μg) were stimulated with Activin A (50 ng/ml) and CR-1ΔC (400 ng/ml) and analyzed by Western blot analysis. (B and C) EpH-4 WT or EpH-4 N cells transfected with pCI-neo (6 μg) (lanes 1, 2, and 4) and ALK4-2 (6, 4, 2, and 1 μg) were stimulated with EGF (50 ng/ml) and CR-1ΔC (200 ng/ml) and analyzed by Western blot analysis. MW, molecular weight in thousands; C, control.

Article Snippet: To ensure equal loading of proteins the blots were stripped and reprobed with Smad-2/Smad-3 (2 μg/ml) (Upstate Biotechnology), total MAPK (1:1,000) (Biolab Laboratories), or total AKT (1:1,000) (Biolab Laboratories) rabbit polyclonal antibodies.

Techniques: Transfection, Dominant Negative Mutation, Plasmid Preparation, Recombinant, Western Blot, Molecular Weight