Disruption of CTNNB1/LEF-1 Complex by Small Molecule Inhibitors Induces Apoptosis in B-CLL Cells in Vitro and in Vivo

Abstract Deregulated WNT signaling has been implicated in variety of tumors. Previous findings suggest critical components of WNT signaling pathway (WNTs, FZD and LEF-1) are overepxressed in B-cell chronic lymphocytic leukemia (CLL) at the m-RNA level. β-catenin (CTNNB1)/LEF-1 transcription activation complex is the key component of WNT signaling which governs the expression of WNT target genes. Critical genes like C-MYC, survivin (SURV), cyclin D1 (CCND1) and LEF-1 which are also overexpressed in CLL are downstream targets of this pathway. In the current study we attempted to inhibit CTNNB1/LEF-1 interaction in CLL cells using the novel small molecule inhibitors CGP049090 and PKF115-584 and observed the survival potential both in vivo and in vitro. Substantial overexpression of LEF-1 and dephospho-CTNNB1, in CLL cells was observed when compared to healthy CD19 positive B cells and PBMCs suggests activation of WNT signaling in CLL. The LC50 calculated from 30 CLL patients was 1.18 μM for CGP049090 and 1.10 μM for PKF115-584 by the ATP based assay. This was confirmed by the DiSC assay LC50 of 0.33 μM (CGP049090) and 0.23 μM (PKF115-584) for primary B-CLL cells (n=3). Both substances showed little effects on normal PBMCs (n=5) as seen from the LC50 values of 90.92 μM and 39.88 μM for CGP049090 and PKF115-584, respectively. Time course experiments indicate activation of caspase 8, 9, 3 & 7, PARP cleavage and paralleled by downregulation of known inhibitors of apoptosis (IAPs) MCL-1, XIAP and BCL-2 leading to apoptosis. Co-immune precipitation of CTNNB1/LEF-1 complex was inhibited in the presence of CGP049090 and PKF115-584 suggesting the specificity of the interaction. Cellular uptake of PKF115-584 and cytoplasmic co-localisation with lef-1 was observed by fluorescence microscopy. In vivo studies reveal that both substances are highly tolerable and effective in eradicating tumor growth in xenografts. The tumor inhibitory rate (IRmax) was found to be 78% and 63.8% for CGP049090 and PKF115-584, respectively. These results indicate that LEF-1 is a promising target for B-CLL therapy as CGP049090 and PKF115-584 effectively induce apoptosis within micromolar concentrations in CLL cells but do not affect healthy cells at these doses. Downregulation of WNT target genes and IAPs hasten the apoptotic mechanism. Moreover, and these inhibitors are highly effective and tolerable in vivo which identifies both compounds as promising drugs for targeted therapy in CLL.

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Faculty Opinions recommendation of Identification of new small molecule inhibitors of cystic fibrosis transmembrane conductance regulator protein: in vitro and in vivo studies.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1069767.522696 ◽

2007 ◽

Author(s):

Kim Barrett

Keyword(s):

Cystic Fibrosis ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

In Vivo Studies ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Regulator Protein ◽

Cystic Fibrosis Transmembrane

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Amygdala-Based Altered miRNome and Epigenetic Contribution of miR-128-3p in Conferring Susceptibility to Depression-Like Behavior via Wnt Signaling

The International Journal of Neuropsychopharmacology ◽

10.1093/ijnp/pyz071 ◽

2020 ◽

Vol 23 (3) ◽

pp. 165-177 ◽

Cited By ~ 5

Author(s):

Bhaskar Roy ◽

Michael Dunbar ◽

Juhee Agrawal ◽

Lauren Allen ◽

Yogesh Dwivedi

Keyword(s):

Wnt Signaling ◽

Target Genes ◽

Rna Binding ◽

Statistical Significance ◽

Neuroblastoma Cell ◽

Wnt Signaling Pathway ◽

Target Prediction ◽

In Vivo Model

Abstract Background Recent studies suggest that microRNAs (miRNAs) can participate in depression pathogenesis by altering a host of genes that are critical in corticolimbic functioning. The present study focuses on examining whether alterations in the miRNA network in the amygdala are associated with susceptibility or resiliency to develop depression-like behavior in rats. Methods Amygdala-specific altered miRNA transcriptomics were determined in a rat depression model following next-generation sequencing method. Target prediction analyses (cis- and trans) and qPCR-based assays were performed to decipher the functional role of altered miRNAs. miRNA-specific target interaction was determined using in vitro transfection assay in neuroblastoma cell line. miRNA-specific findings from the rat in vivo model were further replicated in postmortem amygdala of major depressive disorder (MDD) subjects. Results Changes in miRNome identified 17 significantly upregulated and 8 significantly downregulated miRNAs in amygdala of learned helpless (LH) compared with nonlearned helpless rats. Prediction analysis showed that the majority of the upregulated miRNAs had target genes enriched for the Wnt signaling pathway. Among altered miRNAs, upregulated miR-128-3p was identified as a top hit based on statistical significance and magnitude of change in LH rats. Target validation showed significant downregulation of Wnt signaling genes in amygdala of LH rats. A discernable increase in expression of amygdalar miR-128-3p along with significant downregulation of key target genes from Wnt signaling (WNT5B, DVL, and LEF1) was noted in MDD subjects. Overexpression of miR-128-3p in a cellular model lead to a marked decrease in the expression of Dvl1 and Lef1 genes, confirming them as validated targets of miR-128-3p. Additional evidence suggested that the amygdala-specific diminished expression of transcriptional repressor Snai1 could be potentially linked to induced miR-128-2 expression in LH rats. Furthermore, an amygdala-specific posttranscriptional switching mechanism could be active between miR-128-3p and RNA binding protein Arpp21 to gain control over their target genes such as Lef1. Conclusion Our study suggests that in amygdala a specific set of miRNAs may play an important role in depression susceptibility, which could potentially be mediated through Wnt signaling.

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Small Molecule Inhibitors of Wnt/β-Catenin/Lef-1 Signaling Induces Apoptosis in Chronic Lymphocytic Leukemia Cells In Vitro and In Vivo

Neoplasia ◽

10.1593/neo.91972 ◽

2010 ◽

Vol 12 (4) ◽

pp. 326-IN6 ◽

Cited By ~ 76

Author(s):

Rajesh Kumar Gandhirajan ◽

Peter Anton Staib ◽

Katharina Minke ◽

Iris Gehrke ◽

Günther Plickert ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Lymphocytic Leukemia ◽

Leukemia Cells

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Small-Molecule Inhibitors of the Rce1p CaaX Protease

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107307226 ◽

2007 ◽

Vol 12 (7) ◽

pp. 983-993 ◽

Cited By ~ 43

Author(s):

Surya P. Manandhar ◽

Emily R. Hildebrandt ◽

Walter K. Schmidt

Keyword(s):

Small Molecule ◽

Small Molecule Inhibitors ◽

Proteolytic Processing ◽

In Vivo Studies ◽

In Vitro Assays ◽

Selective Agents ◽

Wild Type Yeast ◽

Micromolar Range

The Rce1p protease is required for the maturation of the Ras GTPase and certain other isoprenylated proteins and is considered a chemotherapeutic target. To identify new small-molecule inhibitors of Rce1p, the authors screened the National Cancer Institute Diversity Set compound library using in vitro assays to monitor the proteolytic processing of peptides derived from Ras and the yeast a-factor mating pheromone. Of 46 inhibitors initially identified with a Ras-based assay, only 9 were effective in the pheromone-based assay. The IC50 values of these 9 compounds were in the low micromolar range for both yeast (6-35 µM) and human Rce1p (0.4-46 µM). Four compounds were somewhat Rce1p selective in that they partially inhibited the Ste24p protease and did not inhibit Ste14p isoprenylcysteine carboxyl methyltransferase, 2 enzymes also involved in the maturation of isoprenylated proteins. The remaining 5 compounds inhibited all 3 enzymes. The 2 most Rce1p-selective agents were ineffective trypsin inhibitors, further supporting the specificity of these agents for Rce1p. The 5 least specific compounds formed colloidal aggregates, a proposed common feature of promiscuous inhibitors. Interestingly, the most specific Rce1p inhibitor also formed a colloidal aggregate. In vivo studies revealed that treatment of wild-type yeast with 1 compound induced a Ras2p delocalization phenotype that mimics observed effects in rce1 ste24 null yeast. The 9 compounds identified in this study represent new tools for understanding the enzymology of postisoprenylation-modifying enzymes and provide new insight for the future development of Rce1p inhibitors. ( Journal of Biomolecular Screening 2007:983-993)

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Identification of New Small Molecule Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Protein: In Vitro and in Vivo Studies

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.30.502 ◽

2007 ◽

Vol 30 (3) ◽

pp. 502-507 ◽

Cited By ~ 15

Author(s):

Chatchai Muanprasat ◽

Santi Kaewmokul ◽

Varanuj Chatsudthipong

Keyword(s):

Cystic Fibrosis ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

In Vivo Studies ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Regulator Protein ◽

Cystic Fibrosis Transmembrane

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Targeting dysregulation signatures of p53-MDM2 interactions to identify newer small molecule inhibitors for cancer therapy: Insight from computational direction

10.21203/rs.3.rs-217761/v1 ◽

2021 ◽

Author(s):

Prosper Obed Chukwuemeka ◽

Haruna Isiyaku Umar ◽

Opeyemi Iwaloye ◽

Oluwaseyi Matthew Oretade ◽

Christopher Busayo Olowosoke ◽

...

Keyword(s):

Binding Affinity ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Therapeutic Option ◽

Pharmacophore Model ◽

In Vivo Studies ◽

Hydrogen Bond Acceptor ◽

Potential Inhibitors

Abstract Dysregulation of the p53-MDM2 interactions has been implicated in majority of human tumors presenting a target for finding small molecule inhibitors. In this study, a training set of 17 experimentally tested inhibitors of MDM2 was used to develop series of pharmacophore models among which a four-featured (AHRR_1) model with one hydrogen bond acceptor, one hydrophobic group and two aromatic ring features and characterized by a survival score of 4.176 was considered significant among the top ranked generated hypothesis. Further, the model was validated by an external set of actives and decoy molecules and was found to exhibit encouraging statistical attributes (such as AUC > 0.7, BEDROC > 0.5 and EF > 1.0 etc). The model was used to screen the ZINC compound database, from the database, the top best 1375 hits satisfying the pharmacophore model was were docked to MDM2 protein to identify the likely interactions of the compounds as well as their binding affinity with MDM2. Further, druglikeness and pharmacokinetic properties screening on top-ranked compounds with higher binding affinity than reference inhibitors revealed four compounds (ZINC02639178, ZINC38933175, ZINC77969611, and ZINC06752762) with suitable pharmacological properties including low ligand toxicity. Investigation of the dynamic behaviour of each candidate inhibitors in complex with MDM2 via molecular dynamic simulation suggested ZINC02639178 and ZINC06752762 as the most potential inhibitors. Thus, these compounds may emerged as therapeutic option for cancer treatment after extensive in vitro and in vivo studies.

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IN SILICO IDENTIFICATION OF APOBEC3B SMALL MOLECULE INHIBITORS FROM DTP-NCI LIBRARIES

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2021v13i3.41600 ◽

2021 ◽

pp. 165-170

Author(s):

MARYAN MOHAMUD MOHAMED ◽

NOR ATIQAH JUSRIL ◽

MOHD ILHAM ADENAN ◽

N. G. KWOK WEN

Keyword(s):

Virtual Screening ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

3D Structure ◽

In Vivo Studies ◽

Amino Acid Residues ◽

Autodock Vina ◽

Raloxifene Hydrochloride

Objective: APOBEC3B (A3B) enzyme causes C-to-T or C-to-G somatic alteration in the cancer genome, leading to the evolution of a broad spectrum of human cancers. The present study aims to identify A3B small molecule inhibitors using a top-down approach via pharmacoinformatic virtual screening. Methods: Virtual screening of 2951 drug-alike molecules with diversified structures from the National Cancer Institute Development Therapeutics Program (DTP-NCI) compounds library was performed using GOLD and AutoDock Vina docking programs against the 3D structure of A3B (PDB ID: 5TD5). Results: Amongst the docked compounds, Nordracorubin, NSC641233 and Raloxifene hydrochloride showed the most potent binding affinities towards A3B on both Autodock/Vina and GOLD. Several significant similarities were observed between A3B and the three hits, including hydrogen bonds and pi-pi stacking. The three compounds also exhibited interaction with the centralized zinc cofactor and amino acid residues that directly contribute the deaminase activity of A3B enzyme. Conclusion: We hypothesize that the findings from this study could significantly shorten the quest for novel molecules against the A3B after confirmation with subsequent in vitro and in vivo studies in the near future.

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Abstract 361: Activation of Wnt Pathway in Macrophages During Atherosclerosis Regression

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.361 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Prashanthi Menon ◽

Yulia Vengrenyuk ◽

Yoscar Ogando ◽

Stephen Ramsey ◽

Elizabeth Gold ◽

...

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Mouse Models ◽

Target Genes ◽

Wnt Signaling Pathway ◽

Macrophage Migration ◽

Canonical Wnt Signaling ◽

Canonical Wnt

Introduction and Objective: Transcriptome analysis of plaque macrophages in two different mouse models of atherosclerosis regression revealed an over representation of consensus binding site sequences for the T-cell factor (TCF)/Lymphoid enhancer binding factor (LEF) family of transcription factors, suggesting canonical Wnt signaling pathway activation during regression in vivo. The canonical Wnt/β-catenin signaling pathway is important for cardiac development and regulates processes such as migration, invasion and tissue repair. However, its function in plaque macrophages is unclear. The objective of the study was to understand the role of canonical Wnt signaling in macrophages during regression using in vivo and in vitro approaches. Methods and Results: Immunohistochemistry of atherosclerotic arterial sections in mouse models of atherosclerosis regression (Reversa and aortic arch transplant) showed a significant increase in β-catenin expression in regressing vs. progressing macrophages. Elevated transcript levels of canonical Wnt downstream targets Ctnnb1, Lrp1 and Gja1 were detected in regressing plaque macrophages isolated by laser capture microdissection (LCM). Canonical Wnt signaling was further investigated in Wnt3a-stimulated primary bone marrow-derived macrophages (BMDM) in vitro, revealing upregulation of pathway target genes Ctnnb1 and Axin2. Furthermore, immunofluorescence analysis of BMDM stimulated with Wnt3a showed increased nuclear expression of β-catenin. Macrophage cell migration evaluated by scratch wound assay revealed a significant increase in migration in Wnt3a-treated vs. untreated BMDM. Conclusions: Our findings demonstrate that canonical Wnt signaling is activated in regressing plaque macrophages and regulates macrophage migration in vitro. Future studies are aimed at understanding the mechanism by which Wnt modulates macrophage migration.

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The microRNA miR-8 is a conserved negative regulator of Wnt signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0807763105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15417-15422 ◽

Cited By ~ 130

Author(s):

Jennifer A. Kennell ◽

Isabelle Gerin ◽

Ormond A. MacDougald ◽

Ken M. Cadigan

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Target Genes ◽

Wnt Signaling Pathway ◽

Negative Regulator ◽

Animal Development ◽

Protein Levels ◽

Evolutionarily Conserved ◽

Multiple Levels

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

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