The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high‐throughput ...screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2O3, and NiO) could interfere with embryo hatching by a chelator‐sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose‐dependent fashion. A peptide‐based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine‐based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species.
Automated high‐throughput screening using zebrafish embryos is used for hazard assessment of 24 representative metal oxide nanoparticles. Four nanoparticles are found to interfere with zebrafish embryo hatching. Hatching interference is a result of toxic metal ion shedding from nanoparticles, compromising the zebrafish hatching enzyme 1 (ZHE1) activity. The structural and functional similarities of hatching enzymes across fish species suggest that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of engineered nanoparticles.
Most mammalian genes produce multiple mRNA isoforms derived from alternative pre-mRNA splicing, with each alternative exon controlled by a complex network of regulatory factors. The identification of ...these regulators can be laborious and is usually carried out one factor at a time. We have developed a broadly applicable high-throughput screening method that simultaneously identifies multiple positive and negative regulators of a particular exon. Two minigene reporters were constructed: One produces green fluorescent protein (GFP) from the mRNA including an exon, and red fluorescent protein (RFP) from the mRNA lacking the exon; the other switches these fluorescent products of exon inclusion and exclusion. Combining results from these two reporters eliminates many false positives and greatly enriches for true splicing regulators. After extensive optimization of this method, we performed a gain-of-function screen of 15,779 cDNA clones and identified 40 genes affecting exon 18 of Discs large homolog 4 (Dlg4; also known as post-synaptic density protein 95 Psd-95). We confirmed that 28 of the 34 recoverable clones alter reporter splicing in RT-PCR assays. Remarkably, 18 of the identified genes encode splicing factors or RNA binding proteins, including PTBP1, a previously identified regulator of this exon. Loss-of-function experiments examining endogenous Dlg4 transcripts validated the effects of five of eight genes tested in independent cell lines, and two genes were further confirmed to regulate Dlg4 splicing in primary neurons. These results identify multiple new regulators of Dlg4 splicing, and validate an approach to isolating splicing regulators for almost any cassette exon from libraries of cDNAs, shRNAs, or small molecules.
Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic ...stem cells (HSCs). Here, we describe small molecule inhibitors of PTPσ that promote HSC regeneration in vivo. Systemic administration of the PTPσ inhibitor, DJ001, or its analog, to irradiated mice promotes HSC regeneration, accelerates hematologic recovery, and improves survival. Similarly, DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPσ and antagonizes PTPσ via unique non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-X
. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTPσ inhibitors for human hematopoietic regeneration.
Background and Purpose
Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, ...activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.
Experimental Approach
Human precision cut lung slices from non‐asthma donors and primary human airway smooth muscle (HASM) cells from both non‐asthma and asthma donors were utilized. Phosphorylation of Akt, myosin phosphatase target subunit 1 (MYPT1) and myosin light chain (MLC) were assessed in HASM cells following either PI3K inhibitor or siRNA treatment. HASM relaxation was assessed by micro‐pattern deformation. Reversal of constriction of airways was assessed following stimulation with PI3K or ROCK inhibitors.
Key Results
Soluble inhibitors or PI3Kδ knockdown reversed carbachol‐induced constriction of human airways, relaxed agonist‐contracted HASM and inhibited pAkt, pMYPT1 and pMLC in HASM. Similarly, inhibition of Rho kinase also dilated human PCLS airways and suppressed pMYPT1 and pMLC. Baseline pMYPT1 was significantly elevated in HASM cells derived from asthma donors in comparison with non‐asthma donors. After desensitization of the β2‐adrenoceptors, a PI3Kδ inhibitor remained an effective dilator. In the presence of IL‐13, dilation by a β agonist, but not PI3K inhibitor, was attenuated.
Conclusion and Implications
PI3Kδ inhibitors act as dilators of human small airways. Taken together, these findings provide alternative approaches to the clinical management of airway obstruction in asthma.
Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel ...disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60-70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/).
Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic ...target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.
Most patients with prostate cancer treated with androgen receptor (AR) signaling inhibitors develop therapeutic resistance due to restoration of AR functionality. Thus, there is a critical need for ...novel treatment approaches. Here we investigate the theranostic potential of hu5A10, a humanized mAb specifically targeting free PSA (
).
LNCaP-AR (LNCaP with overexpression of wildtype AR) xenografts (NSG mice) and
_Hi-
transgenic mice were imaged with
Zr- or treated with
Y- or
Ac-labeled hu5A10; biodistribution and subcellular localization were analyzed by gamma counting, PET, autoradiography, and microscopy. Therapeutic efficacy of
Achu5A10 and
Yhu5A10 in LNCaP-AR tumors was assessed by tumor volume measurements, time to nadir (TTN), time to progression (TTP), and survival. Pharmacokinetics of
Zrhu5A10 in nonhuman primates (NHP) were determined using PET.
Biodistribution of radiolabeled hu5A10 constructs was comparable in different mouse models. Specific tumor uptake increased over time and correlated with PSA expression. Treatment with
Y/
Achu5A10 effectively reduced tumor burden and prolonged survival (
≤ 0.0054). Effects of
Yhu5A10 were more immediate than
Achu5A10 (TTN,
< 0.0001) but less sustained (TTP,
< 0.0001). Complete responses were observed in 7 of 18
Achu5A10 and 1 of 9 mice
Yhu5A10. Pharmacokinetics of
Zrhu5A10 were consistent between NHPs and comparable with those in mice.
Zrhu5A10-PET visualized the NHP-prostate over the 2-week observation period.
We present a complete preclinical evaluation of radiolabeled hu5A10 in mouse prostate cancer models and NHPs, and establish hu5A10 as a new theranostic agent that allows highly specific and effective downstream targeting of AR in PSA-expressing tissue. Our data support the clinical translation of radiolabeled hu5A10 for treating prostate cancer.
Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to ...discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.
Alternative splicing has emerged as a promising therapeutic target in a number of human disorders. However, the discovery of compounds that target the splicing reaction has been hindered by the lack ...of suitable high-throughput screening assays. Conversely, the effects of known drugs on the splicing reaction are mostly unclear and not routinely assessed. We have developed a two-color fluorescent reporter for cellular assays of exon inclusion that can accommodate nearly any cassette exon and minimizes interfering effects from changes in transcription and translation. We used microtubule-associated protein tau (MAPT) exon 10, whose missplicing causes frontotemporal dementia, to test the reporter in screening libraries of known bioactive compounds. These screens yielded several compounds that alter the splicing of the exon, both in the reporter and in the endogenous MAPT mRNA. One compound, digoxin, has long been used in the treatment of heart failure, but was not known to modulate splicing. The positive compounds target different signal transduction pathways, and microarray analysis shows that each compound affects the splicing of a different set of exons in addition to MAPT exon 10. Our results identify currently prescribed cardiotonic steroids as modulators of alternative splicing and demonstrate the feasibility of screening for drugs that alter exon inclusion.
A phenotype recognition model was developed for high throughput screening (HTS) of engineered Nano-Materials (eNMs) toxicity using zebrafish embryo developmental response classified, from ...automatically captured images and without manual manipulation of zebrafish positioning, by three basic phenotypes (i.e., hatched, unhatched, and dead). The recognition model was built with a set of vectorial descriptors providing image color and texture information. The best performing model was attained with three image descriptors (color histogram, representative color, and color layout) identified as most suitable from an initial pool of six descriptors. This model had an average recognition accuracy of 97.40±0.95% in a 10-fold cross-validation and 93.75% in a stress test of low quality zebrafish images. The present work has shown that a phenotyping model can be developed with accurate recognition ability suitable for zebrafish-based HTS assays. Although the present methodology was successfully demonstrated for only three basic zebrafish embryonic phenotypes, it can be readily adapted to incorporate more subtle phenotypes.
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