Wnt Signaling in Zebrafish Fin Regeneration: Chemical Biology Using GSK3b Inhibitors

poster abstractBone growth can be impaired due to disease, such as osteoporosis, and Wnt signaling pathways regulate bone growth. The parathyroid hormone (PTH) is therapeutic for anabolic bone growth (bone building), which activates Wnt signaling, leading to bone growth. GSK3b (glycogen synthetase kinase 3 beta) protein inhibitors activate Wnt signaling, including in bone growth models. Our study utilized a zebrafish model system to study Wnt activated fin regeneration and bone growth. Wnt signaling is the first genetically identified step in fin regeneration, and bony rays are the main differentiated cell type in fins. Thus, zebrafish fin regeneration may be a useful model to study Wnt signaling mediated bone growth. Fin regeneration experiments were conducted using various concentrations of GSK3b inhibitor compound for different treatment periods and regenerative outgrowth was measured at 4 and 7 days post amputation. Experiments revealed continuous low concentration (5-6 nM) treatment to be most effective at increasing regeneration. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. In situ hybridization experiments were performed to examine effects of Gsk3b inhibitor on Wnt responsive gene expression. Initial experiments show temporal and spatial changes on individual gene markers following GSK3b inhibitor treatment. Additionally, confocal microscopy and immunofluorescence labeling data indicated that the Wnt signaling intracellular signal transducer, betacatenin, accumulates throughout Gsk3b inhibitor treated tissues. Finally, experiments are underway to quantify phosphohistone-3 staining in regenerating tissue to measure effects of Gsk3b inhibitor on cell proliferation. Together, these data indicate that bone growth in zebrafish fin regeneration is improved by activating Wnt signaling. Zebrafish Wnt signaling experiments provide good model to study bone growth and bone repair mechanisms, and may provide an efficient drug discovery platform

Effects of GSK3-β Inhibitors on Wnt Signaling in Zebrafish Fin Regeneration: Chemical Biology

In order to develop beneficial drugs for osteoporosis it is important to understand the molecular mechanisms of bone regeneration and define specific regulatory factors. Zebrafish can regenerate damaged tissues, and they prove to be a good model to study bone growth and repair. Previous research showed that GSK3β inhibitor compound at various concentrations and for different treatment periods effectively stimulated fin regeneration. Conducted experiments identified temporal and spatial fluctuations on individual gene markers after GSK3β inhibitor treatment at various concentrations. Recent analyzed data uses the Lilly Research Labs experimental compound LSN 2105786 at 3 nM and 5 nM to stimulate tissue regeneration to determine whether activating Wnt signaling produces cell proliferation and β-catenin translocation to the nucleus for zebrafish bone regeneration. This research has potential to identify mechanism of bone growth and repair, leading to more suitable drugs for patients suffering with osteoporosis.This work was supported by a research grant from Lilly Research Labs. Angelica Brannick and Jennifer Mahin were supported by the IUPUI UROP/CRL program

Deletion of Abi3 gene locus exacerbates neuropathological features of Alzheimer's disease in a mouse model of Aβ amyloidosis

Recently, large-scale human genetics studies identified a rare coding variant in the ABI3 gene that is associated with an increased risk of Alzheimer’s disease (AD). However, pathways by which ABI3 contributes to the pathogenesis of AD are unknown. To address this question, we determined whether loss of ABI3 function affects pathological features of AD in the 5XFAD mouse model. We demonstrate that the deletion of Abi3 locus significantly increases amyloid β (Aβ) accumulation and decreases microglia clustering around the plaques. Furthermore, long-term potentiation is impaired in 5XFAD;Abi3 knockout (“Abi3−/−”) mice. Moreover, we identified marked changes in the proportion of microglia subpopulations in Abi3−/− mice using a single-cell RNA sequencing approach. Mechanistic studies demonstrate that Abi3 knockdown in microglia impairs migration and phagocytosis. Together, our study provides the first in vivo functional evidence that loss of ABI3 function may increase the risk of developing AD by affecting Aβ accumulation and neuroinflammation

Aurora A–Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery