Tight-Binding Small-Molecule Carboxylesterase 2 Inhibitors Reduce Intracellular Irinotecan Activation.

As the primary enzyme responsible for the activatable conversion of Irinotecan (CPT-11) to SN-38, carboxylesterase 2 (CES2) is a significant predictive biomarker toward CPT-11-based treatments for pancreatic ductal adenocarcinoma (PDAC). High SN-38 levels from high CES2 activity lead to harmful effects, including life-threatening diarrhea. While alternate strategies have been explored, CES2 inhibition presents an effective strategy to directly alter the pharmacokinetics of CPT-11 conversion, ultimately controlling the amount of SN-38 produced. To address this, we conducted a high-throughput screening to discover 18 small-molecule CES2 inhibitors. The inhibitors are validated by dose-response and counter-screening and 16 of these inhibitors demonstrate selectivity for CES2. These 16 inhibitors inhibit CES2 in cells, indicating cell permeability, and they show inhibition of CPT-11 conversion with the purified enzyme. The top five inhibitors prohibited cell death mediated by CPT-11 when preincubated in PDAC cells. Three of these inhibitors displayed a tight-binding mechanism of action with a strong binding affinity.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

SALL4B, not targeted by IMiD, is important for SALL4-mediated tumorigenesis.

Oncofetal transcription factor SALL4 is essential for cancer cell survival. 1-5 Recently, several groups reported that immunomodulatory imide drugs (IMiDs) could degrade SALL4 in a proteasome-dependent manner. 6,7 Intriguingly, we observed that IMiDs had no effect on SALL4-positive cancer cells. Further studies demonstrated that IMiDs could only degrade SALL4A, one of the SALL4 isoforms. This finding raises the possibility that SALL4B, the isoform not affected by IMiDs, may be essential for SALL4-mediated cancer cell survival. SALL4B knockdown led to an increase in apoptosis and inhibition of cancer cell growth. SALL4B gain-of-function alone led to liver tumor formation in mice. Our observation that protein degraders can possess isoform-specific effects exemplifies the importance of delineating drug action and oncogenesis at the isoform level to develop more effective cancer therapeutics.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Addressing Compound Reactivity and Aggregation Assay Interferences: Case Studies of Biochemical High-Throughput Screening Campaigns Benefiting from the National Institutes of Health Assay Guidance Manual Guidelines.

Compound-dependent assay interferences represent a continued burden in drug and chemical probe discovery. The open-source National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS) Assay Guidance Manual (AGM) established an "Assay Artifacts and Interferences" section to address different sources of artifacts and interferences in biological assays. In addition to the frequent introduction of new chapters in this important topic area, older chapters are periodically updated by experts from academia, industry, and government to include new technologies and practices. Section chapters describe many best practices for mitigating and identifying compound-dependent assay interferences. Using two previously reported biochemical high-throughput screening campaigns for small-molecule inhibitors of the epigenetic targets Rtt109 and NSD2, the authors review best practices and direct readers to high-yield resources in the AGM and elsewhere for the mitigation and identification of compound-dependent reactivity and aggregation assay interferences.

Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening.

Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional "primed" hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using ∼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements.

Cell size homeostasis is maintained by CDK4-dependent activation of p38 MAPK.

While molecules that promote the growth of animal cells have been identified, it remains unclear how such signals are orchestrated to determine a characteristic target size for different cell types. It is increasingly clear that cell size is determined by size checkpoints-mechanisms that restrict the cell cycle progression of cells that are smaller than their target size. Previously, we described a p38 MAPK-dependent cell size checkpoint mechanism whereby p38 is selectively activated and prevents cell cycle progression in cells that are smaller than a given target size. In this study, we show that the specific target size required for inactivation of p38 and transition through the cell cycle is determined by CDK4 activity. Our data suggest a model whereby p38 and CDK4 cooperate analogously to the function of a thermostat: while p38 senses irregularities in size, CDK4 corresponds to the thermostat dial that sets the target size.

Nuisance compounds in cellular assays.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity ("nuisance compounds") are routinely encountered in cellular assays, including phenotypic and high-content screening assays. Much is known regarding compound-dependent assay interferences in cell-free assays. However, despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer number of targets, but rather nuisance compounds, due to their ability to waste significant resources and erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological settings.

Advancements in Assay Technologies and Strategies to Enable Drug Discovery.

Assays drive drug discovery from the exploratory phases to the clinical testing of drug candidates. As such, numerous assay technologies and methodologies have arisen to support drug discovery efforts. Robust identification and characterization of tractable chemical matter requires biochemical, biophysical, and cellular approaches and often benefits from high-throughput methods. To increase throughput, efforts have been made to provide assays in miniaturized volumes which can be arrayed in microtiter plates to support the testing of as many as 100,000 samples/day. Alongside these efforts has been the growth of microtiter plate-free formats with encoded libraries that can support the screening of billions of compounds, a hunt for new drug modalities, as well as emphasis on more disease relevant formats using complex cell models of disease states. This review will focus on recent developments in high-throughput assay technologies applied to identify starting points for drug discovery. We also provide recommendations on strategies for implementing various assay types to select high quality leads for drug development.

Multimodal small-molecule screening for human prion protein binders.

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mm), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. Whereas orthogonal binder discovery methods could yield high-affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

Systematic Chemogenetic Library Assembly.

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners.

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin. In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

A Scalable Pipeline for High-Throughput Flow Cytometry.

Flow cytometry (FC) provides high-content data for a variety of applications, including phenotypic analysis of cell surface and intracellular markers, characterization of cell supernatant or lysates, and gene expression analysis. Historically, sample preparation, acquisition, and analysis have presented as a bottleneck for running such types of assays at scale. This article will outline the solutions that have been implemented at Novartis which have allowed high-throughput FC to be successfully conducted and analyzed for a variety of cell-based assays. While these experiments were generally conducted to measure phenotypic responses from a well-characterized and information-rich small molecular probe library known as the Mechanism-of-Action (MoA) Box, they are broadly applicable to any type of test sample. The article focuses on application of automated methods for FC sample preparation in 384-well assay plates. It also highlights a pipeline for analyzing large volumes of FC data, covering a visualization approach that facilitates review of screen-level data by dynamically embedding FlowJo (FJ) workspace images for each sample into a Spotfire file, directly linking them to the metric being observed. Finally, an application of these methods to a screen for MHC-I expression upregulators is discussed.

Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays.

Luciferase-based reporter assays are powerful tools for monitoring gene expression in cells because of their ultrasensitive detection capacity and wide dynamic range. Here we describe the characterization and use of a luciferase reporter enzyme derived from the marine copepod Metridia luciferase family, referred to as TurboLuc luciferase (TurboLuc). To develop TurboLuc, the wild-type luciferase was modified to decrease its size, increase brightness, slow luminescent signal decay, and provide for efficient intracellular expression. To determine the enzyme susceptibility to compound inhibition and judge the suitability of using of TurboLuc as a reporter in screening assays, purified TurboLuc enzyme was screened for inhibitors using two different compound libraries. No inhibitors of this enzyme were identified in a library representative of typical diverse low molecular weight (LMW) compounds using a purified TurboLuc enzyme assay supporting that such libraries will show very low interference with this enzyme. We were able to identify a few inhibitors from a purified natural product library which can serve as useful tools to validate assays using TurboLuc. In addition to the inhibitor profile for TurboLuc we describe the use of this reporter in cells employing miniaturized assay volumes within 1536-well plates. TurboLuc luciferase is the smallest luciferase reporter enzyme described to date (16 kDa), shows bright luminescence and low interference by LMW compounds, and therefore should provide an ideal reporter in assays applied to high-throughput screening.

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models.

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

Matrix-Based Activity Pattern Classification as a Novel Method for the Characterization of Enzyme Inhibitors Derived from High-Throughput Screening.

One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4S × 4I matrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC50-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC50-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.

Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase.

Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.

Examining Ligand-Based Stabilization of Proteins in Cells with MEK1 Kinase Inhibitors.

In this study, we describe the evaluation of a cell-based protein stability assay using ß-galactosidase fragment complementation technology performed in two independent laboratories. The assay is based on the ability of certain ligands to bind to a protein leading to a ligand-protein complex that has a different stability than the free protein. The assay employed a prolabeled-tagged MEK1 kinase stably expressed in A549 cells and this was used to evaluate focused sets of compounds containing known MEK1inhibitors as well as a random set of compounds. An assay using a prolabeled-tagged lysine methyltransferase known as G9a expressed in A549 cells was used as a counterscreen. In one study, it was found that the majority of MEK1 inhibitors were either found as inactive (52%) or showed a selective inhibitory response (18%) in the cell-based MEK1 assay; however, eight compounds showed a specific activation response consistent with stabilization of MEK1 in cells. Examination of these stabilizing compounds showed that three of these were analogs of hypothemycin, a known covalent allosteric MEK1 inhibitor, while the remaining compounds covered one structural class. Both laboratories were able to confirm activity in the cell-based MEK1 assay for known MEK1 inhibitors and found that this activity was highly selective over the G9a counterscreen assay. Screening of a mechanism of action library containing compounds with bioactivity annotations against the cell-based MEK1 assay did not reveal any mechanisms leading to an increase in signal other than inhibitors of MEK1. This study supports that the MEK1 cellular protein stability assay is sensitive to certain MEK1 inhibitors, often noncompetitive inhibitors with respect to ATP. The cellular stability assay format could be useful to rapidly filter kinase inhibitor hit lists for allosteric kinase inhibitors and support target engagement in cells.

Structure activity relationships of human galactokinase inhibitors.

Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor.