Douglas S. Auld

Biochemical, Cellular and Biophysical Characterization of a Potent Inhibitor of Mutant Isocitrate Dehydrogenase IDH1

Background: IDH1 R132H, implicated in glioblastoma and AML, produces the oncometabolite 2-HG. Results: A detailed binding mechanism of a small molecule inhibitor (ML309) is proposed. Conclusion: ML309 competes with α-KG but is uncompetitive with NADPH and rapidly and reversibly affects cellular 2-HG levels. Significance: Understanding IDH1 R132H inhibition sets the stage for targeting IDH1 R132H for the treatment of cancer. Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (−) isomer is over 400-fold less active (IC50 = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.

A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation

Background: The calcium-activated chloride channel ANO1 regulates multiple physiological processes. Results: We identified residues that when mutated affected channel activity, intracellular trafficking, or localization and report the first structure-function map of ANO1. Conclusion: The re-entrant loop mediates calcium/voltage sensitivity and activation of ANO1. Significance: We provide new tools for studying ANO1 function in biological systems and its potential as a therapeutic target. The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.

Bioluminescent Assays for Cytochrome P450 Enzymes

The cytochrome P450 (CYP) family contains 57 enzymes in humans. The activity of CYPs against xenobiotics is a primary consideration in drug optimization efforts. Here we describe a series of bioluminescent assays that enable the rapid profiling of CYP activity against compound collections. The assays employ a coupled-enzyme format where firefly luciferase is used to measure CYP enzyme activity through metabolism of pro-luciferase substrates.

Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays

Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment.

Comparison of Compound Administration Methods in Biochemical Assays: Effects on Apparent Compound Potency Using Either Assay-Ready Compound Plates or Pin Tool- Delivered Compounds

Compound sample preparation and delivery are the most critical steps in high-throughput screening (HTS) campaigns. Historically, several methods of compound delivery to assays have been used for HTS, including intermediate plates with prediluted compounds, assay-ready plates (ARPs) using either preplated dried compound films or nanoliter DMSO spots of compounds, as well as pin tool–delivered compounds. We and others have observed differences in apparent compound potency depending on the compound delivery method. To quantitatively measure compound potency differences due to the chosen delivery methods, we conducted a controlled study using a validated biochemical luciferase assay and compared potencies when compounds were delivered in either ARPs (using acoustic dispensed nanoliter spots) or by pin tool. Here we compare hit rates, confirmation rates, false-positive rates, and false-negative rates between the two delivery methods using the luciferase assay. We compared polystyrene (PS) and cyclic olefin copolymer (COC) plates using both delivery methods and examined whether ARPs stored at 4 °C were superior to those stored frozen at −20 °C. The data show that the choice of compound delivery method to the assay has an effect on the apparent IC50’s and that pin tool delivery results in more confirmed hits than preplated compounds, resulting in a lower false-negative rate. However, this effect is minimized through the use of COC plates and by obtaining plates in a “just-in-time” mode. Overall, this report provides guidance on using assay-ready compound plates and has affected the way HTS campaigns are using acoustically dispensed plates in our department.

Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase

The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 μM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 μM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 μM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.

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.

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.

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.