Rational Design of Selective Small-Molecule Inhibitors for β‑Catenin/B-Cell Lymphoma 9 Protein–Protein Interactions

Selective inhibition of α-helix-mediated protein–protein interactions (PPIs) with small organic molecules provides great potential for the discovery of chemical probes and therapeutic agents. Protein Data Bank data mining using the HippDB database indicated that (1) the side chains of hydrophobic projecting hot spots at positions <i>i</i>, <i>i</i> + 3, and <i>i</i> + 7 of an α-helix had few orientations when interacting with the second protein and (2) the hot spot pockets of PPI complexes had different sizes, shapes, and chemical groups when interacting with the same hydrophobic projecting hot spots of α-helix. On the basis of these observations, a small organic molecule, 4′-fluoro-<i>N</i>-phenyl-[1,1′-biphenyl]-3-carboxamide, was designed as a generic scaffold that itself directly mimics the binding mode of the side chains of hydrophobic projecting hot spots at positions <i>i</i>, <i>i</i> + 3, and <i>i</i> + 7 of an α-helix. Convenient decoration of this generic scaffold led to the selective disruption of α-helix-mediated PPIs. A series of small-molecule inhibitors selective for β-catenin/B-cell lymphoma 9 (BCL9) over β-catenin/cadherin PPIs was designed and synthesized. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure–activity relationship studies. This new class of inhibitors can selectively disrupt β-catenin/BCL9 over β-catenin/cadherin PPIs, suppress the transactivation of canonical Wnt signaling, downregulate the expression of Wnt target genes, and inhibit the growth of Wnt/β-catenin-dependent cancer cells

mTOR inhibition reduces the miR-146a HFD phenotype <i>in vivo</i>.

(A) C57BL/6 WT and miR-146a-/- mice were placed on HFD for 4 weeks. After 4 weeks, mice were regularly injected with either rapamycin or vehicle control for the duration of the HFD. Shown is the measure of percent weight over time, based on initial weight. Significance levels are indicated for miR-146a-/- vehicle vs. rapamycin groups. (B) Amount of chow consumed per mouse per day over time of diet. (C) % fat of total body mass at week 1 and week 12 HFD. (D) Weight of WT and miR-146a-/- VAT after 14 weeks of HFD in rapamycin or vehicle groups (n = 8/group). p-values for (A) were calculated using RM-ANOVA, corrected for multiple comparisons using Tukey. p-values for (C-D) were calculated using a two-tailed Student’s t-test *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.</p

miR-146a regulates adipose tissue macrophage gene expression during NCD and HFD.

(A) Representative FACS plots of cells collected from the SVF of 20-week old WT and miR-146a-/- mice fed HFD or NCD. Cells were sorted for CD11b+ F4/80+ markers from live, singlet CD45+ cells. Numbers indicate percent cells in gate. (B) Heat map showing total gene expression of sorted ATMs from WT and miR-146a-/- mice fed NCD or HFD, as measured by RNA-seq. Red indicates 100% similarity in total RNA expression to compared sample; yellow indicates dissimilarity. (C-F) Gene Set Enrichment Analysis plots of miR-146a-/- (red) and WT (blue) mice showing (C) (left) enrichment of the ‘inflammatory response’ in miR-146a-/- mice during NCD, and (right) no statistical enrichment of ‘TNFa signaling via NFkB’ during NCD; (D) enrichment of the ‘inflammatory response’ and ‘TNFa signaling via NFkB’ in miR-146a-/- mice during HFD; (E) no statistical enrichment of ‘PI3K/AKT/mTOR signaling’ or ‘mTORC1 signaling’ during NCD; (F) enrichment of ‘PI3K/AKT/mTOR signaling’ and ‘mTORC1 signaling’ in miR-146a-/- mice during HFD. (g-i) Relative expression of the top hits within (G) the ‘inflammatory response’, (H) ‘PI3K/AKT/mTOR/mTORC1 signaling’, and (I) ‘Reactive oxygen species’ upregulated in miR-146a-/- ATMs compared to WT, as determined by RNA-seq. The WT FPKM averages were set to a relative expression value of 1, with data shown as mean±SD. p-value was calculated using two-tailed Student’s t-test. *pS5 Fig.</p

miR-146a protects against weight and fat gain and liver steatosis during diet-induced obesity.

(A) C57BL/6 WT and miR-146a-/- mice were placed on HFD. Measure of percent weight over time of WT (blue) and miR-146a-/- mice (green), based on initial weight. (B) Body weight (g) of WT and miR-146a-/- mice over time. (C) Percent body fat of WT and miR-146a-/- at time points 5, 8, and 12 weeks HFD. (D) Amount of chow consumed per mouse each week over time of diet. (E) H&E staining of representative sections of VAT (left) and liver (right) at week 14 of diet treatment. miR-146a-/- VAT is hypertrophied and liver has severe steatosis compared to WT. 10x magnification; red line indicates 200 μm. (F) Histology scores of H&E stained liver tissues, measuring steatosis and inflammatory foci within livers at 14 weeks HFD. (G) Weight of VAT at 14 weeks HFD. (H) Average respiratory exchange ratio of mice on HFD for 0, 3, or 18 weeks, calculated by VO2 and VCO2 max levels, measured at day and night. (I) Average XY movement of mice, measured at day and night. (J) Average energy expenditure by mice in kcal/hour, measured at day and night. Data are shown as mean ± SEM (n = 6), 5 repeats were performed for the HFD experiments assessing weight gain and adiposity. p-value was calculated using two-tailed Student’s t-test. *pS1 Fig.</p

The miR-146a target Traf6 downregulates inflammation and alters cellular metabolism in activated macrophages.

(A) FPKM values of Targetscan-predicted miR-146a-5p target Traf6 in WT or miR-146a-/- ATMs during HFD, as determined by RNA-seq. (B) Western blot for Traf6 and beta-actin in lysates from WT (1 and 2) or miR-146a-/- (3 and 4) BMDMs double-stimulated with media control or LPS. (C) Western blot for Traf6 and GAPDH in lysates from Traf6 CRISPR and Empty Vector control RAW cells stimulated with media control or LPS. (D) mRNA expression of il6, ccl2, and il12 in Traf6 Cr and EV (empty vector) control RAW cells. (E) mRNA expression of Il6, Ccl2, and Il12 in WT or miR-146a-/- BMDMs treated with media, LPS, Traf6i, or Traf6i+LPS. (F) mRNA expression of metabolic genes Hif1a, Hk2, and Adora2b in WT or miR-146a-/- BMDMs double stimulated with media, LPS, Traf6i, or Traf6i+LPS. (G) OCR of WT and miR-146a-/- BMDMs double-stimulated with media, LPS, Traf6i, or Traf6i+LPS, followed by Seahorse analysis with addition of glucose (gluc), oligomycin (OM), FCCP, and rotenone (rot)/antimycin A (AA). (H) OCR of Traf6 Cr and EV control RAW cells double-stimulated with LPS or media, followed by addition of glucose (gluc), OM, FCCP, and Rot/AA. Data are shown as mean±SEM (d-f) or as individual mice (a-b). p-values were calculated using two-tailed Student’s t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.</p

miR-146a constrains inflammation and is expressed in adipose tissue.

(A-C) qRT-PCR expression levels of various mRNAs (shown on x axis) measured in WT (black) or miR-146a (grey) mice in whole VAT at (A) 0 weeks HFD; (B) 14 weeks on NCD; and (C) 14 weeks on HFD. (D-F) qRT-PCR expression levels of various mRNAs (shown on x axis) measured in WT or miR-146a mice in liver at (D) 0 weeks HFD; (E) 14 weeks on NCD; and (F) 14 weeks on HFD. (G) Western blot for P-IKBa, total IKBa, and GAPDH in lysates of whole VAT collected from 20-week old WT or miR-146a-/- mice fed NCD or HFD. (H) H&E staining of VAT of WT and miR-146a-/- mice fed HFD for 18 weeks. Black arrows indicate areas of inflammation. Two representative examples from miR-146a-/- VAT are shown, with one representative WT mouse; 400x magnification. (I) Mature miR-146a expression relative to 5s rRNA, measured in the SVF and adipocyte fraction of WT and miR-146a-/- mice fed NCD. (J-K) Ptprc (CD45) and Leptin expression levels, relative to L32, were measured as controls for fractionation purity. Samples were normalized by setting WT expression to 1. Data are shown as mean ± SEM (a-f) (n = 10) or as individual mice (h-k) (n = 5); lysates from individual mice (g) (n = 2). p-value was calculated using two-tailed Student’s t-test. *pS4 Fig.</p

miR-146a is required for protection from a diabetic phenotype during diet-induced obesity.

All measurements were performed in WT (blue) and miR-146a-/- (green) mice at 0, 3, and 18 weeks HFD. (A) GTTs measuring blood glucose levels, following injection of glucose at 0 minutes and measured over time for 120 minutes. (B) Area under curve of GTTs. (C) Serum insulin levels, measured via ELISA. (D) HOMA-β levels, measuring beta cell function. (E) HOMA-IR levels, measuring insulin resistance. Data are shown as mean ± SEM (a) or as individual mice (b-e) (n = 5). p-value was calculated using two-tailed Student’s t-test. *pS3 Fig.</p