Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening.

Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine.

Drug Library Screening for the Identification of Ionophores That Correct the Mistrafficking Disorder Associated with Oxalosis Kidney Disease.

Primary hyperoxaluria is the underlying cause of oxalosis and is a life-threatening autosomal recessive disease, for which treatment may require dialysis or dual liver-kidney transplantation. The most common primary hyperoxaluria type 1 (PH1) is caused by genetic mutations of a liver-specific enzyme alanine:glyoxylate aminotransferase (AGT), which results in the misrouting of AGT from the peroxisomes to the mitochondria. Pharmacoperones are small molecules with the ability to modify misfolded proteins and route them correctly within the cells, which may present an effective strategy to treat AGT misrouting in PH1 disorders. We miniaturized a cell-based high-content assay into 1536-well plate format and screened ~4200 pharmacologically relevant compounds including Food and Drug Administration, European Union, and Japanese-approved drugs. This assay employs CHO cells stably expressing AGT-170, a mutant that predominantly resides in the mitochondria, where we monitor for its relocation to the peroxisomes through automated image acquisition and analysis. The miniaturized 1536-well assay yielded a Z' averaging 0.70 ± 0.07. Three drugs were identified as potential pharmacoperones from this pilot screen, demonstrating the applicability of this assay for large-scale high-throughput screening.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases.

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M. tuberculosis.

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38 350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional ΔAPSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.

Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 µM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

Development of a phenotypic high-content assay to identify pharmacoperone drugs for the treatment of primary hyperoxaluria type 1 by high-throughput screening.

Primary hyperoxaluria is a severe disease for which the best current therapy is dialysis or organ transplantation. These are risky, inconvenient, and costly procedures. In some patients, pyridoxine treatment can delay the need for these surgical procedures. The underlying cause of particular forms of this disease is the misrouting of a specific enzyme, alanine:glyoxylate aminotransferase (AGT), to the mitochondria instead of the peroxisomes. Pharmacoperones are small molecules that can rescue misfolded proteins and redirect them to their correct location, thereby restoring their function and potentially curing disease. In the present study, we miniaturized a cell-based assay to identify pharmacoperone drugs present in large chemical libraries to selectively correct AGT misrouting. This assay employs AGT-170, a mutant form of AGT that predominantly resides in the mitochondria, which we monitor for its relocation to the peroxisomes through automated image acquisition and analysis. Over the course of a pilot screen of 1,280 test compounds, we achieved an average Z'-factor of 0.72±0.02, demonstrating the suitability of this assay for HTS.

Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening.

Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.

Casein kinase 1δ-dependent Wee1 protein degradation.

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.

A FluoPol-ABPP PAD2 high-throughput screen identifies the first calcium site inhibitor targeting the PADs.

The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 µM), to preferentially bind the apoenzyme.

Small-molecule proteostasis regulators for protein conformational diseases.

Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting this mechanism as a promising therapeutic approach. We describe the results of a screen comprised of ∼900,000 small molecules that identified new classes of small-molecule proteostasis regulators that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. These beneficial effects to proteome stability are mediated by HSF-1, FOXO, Nrf-2 and the chaperone machinery through mechanisms that are distinct from current known small-molecule activators of the heat shock response. We suggest that modulation of the proteostasis network by proteostasis regulators may be a promising therapeutic approach for the treatment of a variety of protein conformational diseases.

Inhibition of adrenocortical carcinoma cell proliferation by steroidogenic factor-1 inverse agonists.

CONTEXT: Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). OBJECTIVE: Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). MAIN OUTCOME MEASURES: Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and reverse transcription quantitative PCR were used. RESULTS: SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. CONCLUSION: SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.