Small-molecule inhibitor targeting orphan nuclear receptor COUP-TFII for prostate cancer treatment.

The orphan nuclear receptor COUP-TFII is expressed at a low level in adult tissues, but its expression is increased and shown to promote progression of multiple diseases, including prostate cancer, heart failure, and muscular dystrophy. Suppression of COUP-TFII slows disease progression, making it an intriguing therapeutic target. Here, we identified a potent and specific COUP-TFII inhibitor through high-throughput screening. The inhibitor specifically suppressed COUP-TFII activity to regulate its target genes. Mechanistically, the inhibitor directly bound to the COUP-TFII ligand-binding domain and disrupted COUP-TFII interaction with transcription regulators, including FOXA1, thus repressing COUP-TFII activity on target gene regulation. Through blocking COUP-TFII's oncogenic activity in prostate cancer, the inhibitor efficiently exerted a potent antitumor effect in xenograft mouse models and patient-derived xenograft models. Our study identified a potent and specific COUP-TFII inhibitor that may be useful for the treatment of prostate cancer and possibly other diseases.

A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype.

The RAS proteins are the most frequently mutated oncogenes in cancer, with highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks. Thus, significant efforts have been directed at identifying downstream targets on which RAS is dependent. These efforts have proven challenging, in part due to confounding factors such as reliance on two-dimensional adherent monolayer cell cultures that inadequately recapitulate the physiologic context to which cells are exposed in vivo. To overcome these issues, we implemented a high-throughput screening (HTS) approach using a spheroid-based 3-dimensional culture format, thought to more closely reflect conditions experienced by cells in vivo. Using isogenic cell pairs, differing in the status of KRAS, we identified Proscillaridin A as a selective inhibitor of cells harboring the oncogenic KRasG12V allele. Significantly, the identification of Proscillaridin A was facilitated by the 3D screening platform and would not have been discovered employing standard 2D culturing methods.

A Cytotoxic Three-Dimensional-Spheroid, High-Throughput Assay Using Patient-Derived Glioma Stem Cells.

Glioblastoma (GBM) is the most aggressive primary brain cancer with an average survival time after diagnosis of only 12-14 months, with few (<5%) long-term survivors. A growing body of work suggests that GBMs contain a small population of glioma stem cells (GSCs) that are thought to be major contributors to treatment resistance and disease relapse. Identifying compounds that modulate GSC proliferation would provide highly valuable molecular probes of GSC-directed signaling. However, targeting GSCs pharmacologically has been challenging. Patient-derived GSCs can be cultured as neurospheres, and in vivo these cells functionally recapitulate the heterogeneity of the original tumor. Using patient-derived GSC-enriched cultures, we have developed a 1536-well spheroid-based proliferation assay and completed a pilot screen, testing ~3300 compounds comprising approved drugs. This cytotoxic and automation-friendly assay yielded a signal-to-background (S/B) ratio of 161.3 ± 7.5 and Z' of 0.77 ± 0.02, demonstrating its robustness. Importantly, compounds were identified with anti-GSC activity, demonstrating the applicability of this assay for large-scale high-throughput screening (HTS).

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.

A High-Throughput Time-Resolved Fluorescence Energy Transfer Assay to Screen for Modulators of RGS7/Gß5/R7BP Complex.

G protein-coupled receptors (GPCRs) are excellent drug targets exploited by majority of the Food and Drug Administration-approved medications, but when modulated, are often accompanied by significant adverse effects. Targeting of other elements in GPCR pathways for improved safety and efficacy is thus an unmet need. The strength of GPCR signaling is tightly regulated by regulators of G protein signaling (RGS) proteins, making them attractive drug targets. We focused on a prominent RGS complex in the brain consisting of RGS7 and its binding partners Gß5 and R7BP. These complexes play critical roles in regulating multiple GPCRs and essential physiological processes, yet no small molecule modulators are currently available to modify its function. In this study, we report a novel high-throughput approach to screen for small molecule modulators of the intramolecular transitions in the RGS7/Gß5/R7BP complex known to be involved in its allosteric regulation. We developed a time-resolved fluorescence energy transfer-based in vitro assay that utilizes full-length recombinant proteins and shows consistency, excellent assay statistics, and high level of sensitivity. We demonstrated the potential of this approach by screening two compound libraries (LOPAC 1280 and MicroSource Spectrum). This study confirms the feasibility of the chosen strategy for identifying small molecule modulators of RGS7/Gß5/R7BP complex for impacting signaling downstream of the GPCRs.

Improved Scalability of Neuron-Based Phenotypic Screening Assays for Therapeutic Discovery in Neuropsychiatric Disorders.

There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders.

High-Throughput Screening for Protein Synthesis Inhibitors Targeting Aminoacyl-tRNA Synthetases.

Aminoacylation has been implicated in a wide variety of cancers. Aminoacyl-tRNA synthetases (ARSs) exist in large excess in tumor cells due to their increased demand for translation, whereas most other protein-synthesis apparatuses are quantitatively limited. Among other components that constitute the translation machinery-namely, tRNA, amino acid, ATP, and ARS-ARS is the only target that can be blocked by small molecules. No constitutively active ARSs have been reported, and mutations of ARS can cause inaccurate substrate recognition and malformation of the multi-ARS complex (MSC). Hence, interference of the activity is expected to be independent of genotype without developing resistance. Here, we report a high-throughput screening (HTS) system to find mammalian ARS inhibitors. The rabbit-reticulocyte lysate we used closely resembles both the individual and complexed structures of human ARSs, and it may predispose active compounds that are readily applicable for humankind. This assay was further validated because it identified familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for our purpose. The assay demonstrated excellent quality control (QC) parameters and reproducibility, and is proven ready for further HTS campaigns with large chemical libraries.

A 1536-Well 3D Viability Assay to Assess the Cytotoxic Effect of Drugs on Spheroids.

Evaluation of drug cytotoxicity traditionally relies on use of cell monolayers, which are easily miniaturized to the 1536-well plate format. Three-dimensional (3D) cell culture models have recently gained popularity thanks to their ability to better mimic the complexity of in vivo systems. Despite growing interest in these more physiologically relevant and highly predictive cell-based models for compound profiling and drug discovery, 3D assays are currently performed in a medium- to low-throughput format, either in 96-well or 384-well plates. Here, we describe the design and implementation of a novel high-throughput screening (HTS)-compatible 1536-well plate assay that enables the parallel formation, size monitoring and viability assessment of 3D spheroids in a highly consistent manner. Custom-made plates featuring an ultra-low-attachment surface and round-bottom wells were evaluated for their compatibility with HTS requirements through a luminescence-based cytotoxicity pilot screen of ~3300 drugs from approved drug and National Cancer Institute (NCI) collections. As anticipated, results from this screen were significantly different from a parallel screen performed on cell monolayers. With the ability to achieve an average Z' factor greater than 0.5, this automation-friendly assay can be implemented to either profile lead compounds in a more economical plate format or to interrogate large compound libraries by ultra-HTS (uHTS).

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.

Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate.

ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics.

An Automated Miniaturized Method to Perform and Analyze Antimicrobial Drug Synergy Assays.

In the light of emerging antibiotic resistance mechanisms found in bacteria throughout the world, discovery of drugs that potentiate the effect of currently available antibiotics remains an important aspect of pharmaceutical research in the 21st century. Well-established clinical tests exist to determine synergy in vitro, but these are only optimal for low-throughput experimentation while leaving analysis of results and interpretation of high-throughput microscale assays poorly standardized. Here, we describe a miniaturized broth microdilution checkerboard assay and data analysis method in 384-well plate format that conforms to the Clinical Laboratory and Standards Institute (CLSI) methods. This method has been automated and developed to rapidly determine the synergism of current antibiotics with various beta-lactamase inhibitors emerging from our antimicrobial research efforts. This technique increases test throughput and integrity of results, and saves test compound and labor. We facilitated the interpretation of results with an automated analysis tool allowing us to rapidly qualify inter- and intraplate robustness, determine efficacy of multiple antibiotics at the same time, and standardize the results of synergy interpretation. This procedure should enhance high-throughput antimicrobial drug discovery and supersedes former techniques.

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.

Endogenous and Synthetic ABHD5 Ligands Regulate ABHD5-Perilipin Interactions and Lipolysis in Fat and Muscle.

Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), α-ß hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here, we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 without PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated that ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands, including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.

Application of Parallel Multiparametric Cell-Based FLIPR Detection Assays for the Identification of Modulators of the Muscarinic Acetylcholine Receptor 4 (M4).

Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer's disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M(4) (M(4)), we developed a homogenous, multiparametric, 1536-well assay to measure M(4) receptor agonism, positive allosteric modulation (PAM), and antagonism in a single well. This assay yielded a Z' of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode. Parallel screening of the M(1) and M(5) subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M(4) selective primary agonists, 1450 PAMs, and 2389 antagonist hits. Concentration-response analysis identified 25 selective agonists, 4 PAMs, and 41 antagonists. This demonstrates the advantages of this approach to rapidly identify selective receptor modulators while efficiently removing assay artifacts and undesirable compounds.

Identification of histone deacetylase inhibitors with benzoylhydrazide scaffold that selectively inhibit class I histone deacetylases.

Inhibitors of histone deacetylases (HDACi) hold considerable therapeutic promise as clinical anticancer therapies. However, currently known HDACi exhibit limited isoform specificity, off-target activity, and undesirable pharmaceutical properties. Thus, HDACi with new chemotypes are needed to overcome these limitations. Here, we identify a class of HDACi with a previously undescribed benzoylhydrazide scaffold that is selective for the class I HDACs. These compounds are competitive inhibitors with a fast-on/slow-off HDAC-binding mechanism. We show that the lead compound, UF010, inhibits cancer cell proliferation via class I HDAC inhibition. This causes global changes in protein acetylation and gene expression, resulting in activation of tumor suppressor pathways and concurrent inhibition of several oncogenic pathways. The isotype selectivity coupled with interesting biological activities in suppressing tumor cell proliferation support further preclinical development of the UF010 class of compounds for potential therapeutic applications.

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.

Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and ß.

Zinc metalloproteinases meprin α and meprin ß are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools, we have developed high throughput screening assays to enable discovery of inhibitors of both meprin α and meprin ß and screened a collection of well characterized pharmaceutical agents (library of pharmaceutically active compounds, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin ß. Kinetic studies revealed competitive (PPNDS) and mixed competitive/noncompetitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin ß active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin ß reported to the date. These results demonstrate the ability of meprin α and ß assays to identify selective compounds and discard artifacts of primary screening.