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Abstract

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose–response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction–oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a “tough target” to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

Keywords

ultra-high-throughput screening enzyme assays enzyme kinetics fluorescence methods automation robotics

Introduction

Fumarate hydratase (FH) is an important metabolic enzyme in the Krebs cycle, which reversibly catalyzes the conversion of fumarate to malate (hydration). It is encoded by the FH gene, which produces two isoforms of protein by alternative splicing, a cytosolic form and a mitochondrial form. The mitochondrial form of FH contains an N-terminal mitochondrial targeting sequence, which is removed in the mitochondrion to generate the same protein as that in the cytoplasm. Despite decades of research, a detailed understanding of the mechanism of FH’s biochemical activity remains a matter of debate. The active enzyme is a homo-tetramer in which each active site is made up of residues at the interface of three of the four subunits.¹ FH participates in adenosine triphosphate (ATP) production in mitochondria and the metabolism of amino acids and fumarate in cytoplasm.

There are more than 100 known mutations in the human FH gene. The most common type is missense (57%), followed by frameshifts and nonsense (27%), and other mutations.² Mutations of the FH gene lead to defects of the enzymatic activity either by indirectly compromising the integrity of the protein’s core architecture or by directly affecting residues within the active site that regulate catalytic activity of the enzyme.³ FH gene mutations have been mainly associated with two heritable diseases: fumarate hydratase deficiency (FHD) and hereditary leiomyomatosis and renal cell cancer (HLRCC). Homozygous germline mutations in the FH gene cause autosomal recessive FHD syndrome, which is characterized by early-onset hypotonia, profound psychomotor retardation, seizures, facial dysmorphism, and brain abnormalities.⁴ Mutations of the FH gene severely impair the enzyme activity, which leads to the defect of energy production and the accumulation of fumarate, which is believed to be the cause of clinical symptoms.⁵ Heterozygous germline mutations in the FH gene predispose individuals to HLRCC, characterized by benign leiomyomas of the skin and the uterus and early-onset type II papillary renal cell carcinoma.⁶ In affected individuals, loss of heterozygosity (LOH) in the wild-type allele by somatic mutations leads to severe reduction or absence of FH activity, which is followed by an increased intracellular level of fumarate. Accumulated fumarate acts as an oncometabolite to induce tumorigenesis through competitively inhibiting α-ketoglutarate–dependent dioxygenase enzymes and posttranslationally modifying proteins by succination.^7,8 Aside from FH deficiency and HLRCC, studies have also shown that FH is involved in hypertension, type 2 diabetes, and diabetic kidney disease.^9
–11 For example, deletion of FH in mouse pancreatic β cells leads to progressive glucose intolerance and diabetes after 6–8 weeks. In humans, the fumarate level is higher in islets from donors with type 2 diabetes than from normal donors, and high glucose produces no further increase of fumarate.¹¹

Rescue of defected fumarase activity by re-expression of a normal FH gene could restore the fumarate level and respiratory function of mitochondria,^12,13 which suggests that FH is a potential therapeutic target. The activators of fumarase would enhance the conversion of fumarate to malate, which may rescue the defect of FH. Currently, there are no biochemically characterized activators or inhibitors of FH. Two series of inhibitors have been reported to inhibit the activity of human¹⁴ or Mycobacterium tuberculosis FH.¹⁵ In an attempt to identify nutrient-dependent cytotoxic compounds, Takeuchi et al.¹⁴ screened a collection of 6000 small molecules and discovered a new class of inhibitors of human FH (compounds 1–3 with pyrrolidinone structure). In another report, Kasbekar¹⁵ reported that two inhibitors (compounds 7 and 8) were identified for M. tuberculosis fumarate hydratase (tbFH) after screening a collection of 479,984 small molecules.

To identify small-molecule activators or inhibitors of human FH, we developed a fluorescence-based FH assay by coupling to malate dehydrogenase (MDH)–diaphorase–resazurin and optimized the assay for quantitative high-throughput screening (qHTS) in 1536-well plates. We proceeded to screen a collection of small-molecule libraries to identify new activators or inhibitors of human FH. A total of 57,037 small-molecule compounds were screened, and the majority of hits were triaged by MDH counterscreen assay. One series of the activators for human FH was confirmed and validated in the current study.

Materials and Methods

Reagents and Chemical Libraries

Recombinant human FH (with 6 histidine tag in the N-terminal of protein), dithiothreitol (DTT), and Hank’s Balanced Salt Solution (HBSS) were purchased from Thermo Fisher Scientific (Waltham, MA). L-malate dehydrogenase from pig heart, diaphorase from Clostridium kluyveri, β-NAD (NAD+), resazurin, Brij 35, sodium fumarate dibasic, malic acid, and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St. Louis, MO). Fumarate hydratase-IN-2 was purchased from MedChem Express (Monmouth Junction, NJ). Compound 8 was purchase from Enamine (Monmouth Junction, NJ). Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) was purchased from Cayman Chemical (Ann Arbor, MI). Six libraries were screened: the Sytravon [a retired pharma screening collection that contains a diversity of novel small molecules; from the National Center for Advancing Translational Sciences (NCATS)], NPACT (NCATS Pharmacologically Active Chemical Toolbox; from NCATS), NPC (NCATS Pharmaceutical Collection; NCATS),¹⁶ MIPE (Mechanism Interrogation Plate; NCATS), a kinase inhibitor library (an in-house collection of kinase inhibitors; NCATS), and LOPAC¹²⁸⁰ (Library of Pharmacologically Active Compounds; 1280 pharmacologically active compounds; Sigma-Aldrich).

High-Throughput Screen and Counterscreen

For the primary high-throughput screen, 3 µL of FH solution (containing 13.33 nM human FH, 13.33 IU/mL malic dehydrogenase, 0.2 mM NAD+, 0.067 mg/mL diaphorase, and 0.067 mM resazurin) in the assay buffer (50 mM Tris pH 8.0, 5 mM MgCl₂, and 0.01% Brij 3) was dispensed into each well in a black solid-bottom 1536-well assay plate (Greiner Bio-One, Monroe, NC) using a BioRAPTR FRD (Flying Reagent Dispenser; Beckman Coulter, Brea, CA). A 1536-well Pintool dispenser outfitted with 20 nL pins (Wako Automation, San Diego, CA) was used to transfer 20 nL of DMSO-solubilized compound (cherrypick plates) to each 1536-well assay plate. Each compound was screened at five concentrations: 15 nM, 151 nM, 1.5 µM, 15.4 µM, and 76.9 µM. Following compound transfer, plates were incubated at room temperature (RT) for 10 min. To initiate the reaction, 1 µL of substrate solution containing fumaric acid (160 µM) was dispensed via BioRAPTR FRD. Plates were immediately transferred to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and any resulting resorufin fluorescence was measured (excitation/emission, 525/598 nm) at 0 and 15 min. The exposure time is 1 s. Fluorescence from each well was normalized using enzyme-free and DMSO-treated control wells on each plate, and changes in fluorescence (∆RFU) were calculated using the difference in fluorescent signal for each well at 15 min versus 0 min.

The MDH counterscreen was adapted from the primary high-throughput screen protocol. Three microliters of MDH solution [containing 13.33 IU/ml malic dehydrogenase, 0.2 mM NAD+, 0.067 mg/mL diaphorase, and 0.067 mM resazurin in assay buffer (50 mM Tris pH 8.0, 5 mM MgCl2, and 0.01% Brij 3)] were dispensed in each well in the 1536-well plate. Plates were incubated at RT for 10 min after compound transfer. To initiate the reaction, 1 µL of substrate solution containing malic acid (160 µM) was dispensed. Fluorescence was measured (ex540, em590 nm) at 0 and 5 min. The fluorescence was normalized using enzyme-free and DMSO-treated control wells on each plate, and ∆RFU was calculated using the difference in fluorescent signal for each well at 5 min versus 0 min.

The Reduction–Oxidation (Redox) Assay

The Amplex Red assay was used to detect the redox activity of small molecules. Briefly, 2.5 µL HBSS solution (1.26 mM CaCl2, 0.49 mM MgCl2, and 1 g/L D-glucose) was dispensed into each well in a black solid-bottom 1536-well assay plate. Twenty nanoliters of compounds were pinned into the plate, and the background fluorescence (RFU_0min) was measured in the ViewLux (excitation/emission, 525/598 nm). Two and a half microliters freshly made 2× Amplex Red solution (100 µM Amplex Red, 200 µM DTT, and 2 U/mL HRP in 1× HBSS solution) were added to each well. After 15 min incubation at RT, fluorescence (RFU_15min) was measured in the ViewLux with the same setting. Redox activity of each compound was calculated using corrected fluorescence values (ΔRFU = RFU_15min − RFU_0min). The negative control was DMSO, and the positive controls were walrycin B and chlorinal.

Microscale Thermophoresis (MST)

Recombinant human FH, which contains a polyhistidine tag at the C-terminus, was labeled with Monolith His-Tag Labeling Kit RED-tris-NTA kit (NanoTemper Technologies, Munich, Germany). Briefly, the protein was diluted to 200 nM in a phosphate-buffered saline and Tween (PBS-T) buffer (137 mM NaCl, 2.5 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and pH 7.4 in 0.05% Tween-20). The RED-tris-NTA dye was also diluted in PBS-T to 100 nM. The diluted protein and dye were mixed in a 1:1 volume ratio and incubated for 30 min at RT. After centrifuge 15,000 ×g for 10 min at 4 °C, the supernatant was transferred to a new tube and ready to use.

Compounds were serially diluted in DMSO (16 points, 1:2 dilutions, from 10 mM), and then further diluted 20 times in PBS-T buffer. Ten microliters of labeled protein and 10 µL diluted compounds were mixed and incubated at RT for 2–3 min. The samples were loaded in the standard treated capillaries and measured in a NanoTemper Monolith NT.115 LabelFree instrument (NanoTemper Technologies). The samples were measured at light-emitting diode (LED) power of 40%, and MST power of 40% and 60%. A laser-on time of 30 s and a laser-off time of 5 s were used in this experiment. Curve fitting was performed using GraphPad Prism 5 (La Jolla, CA).

In Vitro ADME Studies

The physicochemical and pharmacokinetics properties of compounds were measured using the following high-throughput in vitro assays: rat microsomal stability assay, parallel artificial membrane permeability assay (PAMPA), and aqueous kinetic solubility assays. The details of these assays are described in a previous study.¹⁷

qHTS Data Analysis

Analysis of compound concentration–response data was performed as previously described.¹⁸ Briefly, raw plate reads for each titration point were first normalized relative to the no-enzyme control and DMSO-only wells as follows: % Activity = [(V_compound – V_DMSO) / (V_DMSO – V_{no enzyme})] × 100, where V_compound denotes the compound well values, V_{no enzyme} denotes the median values of the no-enzyme control wells, and V_DMSO denotes the median values of the DMSO-only wells; and then corrected by applying an NCATS Chemical Genomics Center (NCGC) in-house pattern correction algorithm using compound-free control plates (i.e., DMSO-only plates) at the beginning and end of the compound plate stack.¹⁹ Concentration–response titration points for each compound were fitted to a four-parameter Hill equation²⁰ yielding concentrations of half-maximal activity (AC₅₀) and maximal response (efficacy) values. Compounds were designated as Classes 1–4 according to the type of concentration–response curve observed.^18,21 Curve classes are heuristic measures of data confidence, classifying concentration–responses on the basis of efficacy, the number of data points observed higher than background activity, and the quality of fit. Compounds with curve classes 1.1, 1.2, 2.1, 2.2 (activators) and −1.1, −1.2, −2.1, or −2.2 (inhibitors) were considered active. Class 4 compounds were considered inactive. Compounds with other curve classes were deemed inconclusive. Active compounds were cherrypicked for follow-up experiments.

Results

Assay Design, Optimization, and Miniaturization

To identify activators or inhibitors of human FH, we developed a fluorescence-based FH assay, adapted from a previous study.¹⁵ In our assay format, the FH reaction is coupled to two sequential enzymatic reactions: MDH and diaphorase reactions. In the first coupled MDH reaction, L-malate produced by FH is converted into oxaloacetate, removing L-malate from the system and driving the FH forward reaction. The reduced form of nicotinamide adenine dinucleotide (NADH) generated by MDH is used in the second coupled reaction by diaphorase to convert resazurin to resorufin ( Fig. 1A ), with the further benefit of regenerating NAD⁺ to drive the MDH biochemical reaction. Resorufin is strongly fluorescent, with an excitation wavelength at 530–540 nm and emission at 585–595 nm.^22,23 Therefore, FH activity can be evaluated by measuring fluorescence signals of resorufin.

Figure 1.

Validation of the fumarate hydratase (FH) primary assay and malate dehydrogenase (MDH) counterassay in quantitative high-throughput screening (qHTS). (A) Schematic diagram of the fluorescence-based high-throughput screening assay used to evaluate FH activity. The FH enzymatic reaction is coupled to MDH and the diaphorase reaction for the detection of resorufin fluorescence. (B) Time course of the FH assay with varying concentrations of FH enzyme (0, 5, 10, and 20 nM). Fluorescence was continuously measured every minute for 30 min. The red dotted line indicates the time the signals were measured in the final HTS assay. (C) Km value of fumarate for the FH enzyme. Reactions were assembled with 10 nM FH, the indicated concentration of fumarate, and incubated at room temperature for 15 min. (D) Time course of the MDH assay with varying concentrations of the MDH enzyme (0, 5, 10, and 20 IU/ml). Fluorescence was continuously measured every minute for 30 min. The red dotted line indicates the time the signals were measured. (E) Km value of malate for the MDH enzyme. Reactions were assembled with 10 IU/ml MDH, the indicated concentration of malate, and incubated at room temperature for 5 min. (F) Scatterplot of the percentage of FH activity from a 1536-well plate to validate the assay. Columns 1 and 2 were DMSO positive controls, and columns 3 and 4 were no-FH-enzyme negative controls. In this plate, the signal-to-background (S/B) ratio was 13.8, coefficient of variation (CV) was 6.1, and Z factor was 8.3.

To optimize assay conditions, the initial linear velocity of the enzymatic reaction was determined in the presence of excess fumarate. We found that the enzymatic reaction was linear during a period of 30 min at a concentration of up to 20 nM FH enzyme ( Fig. 1B ). We selected a concentration of 10 nM FH enzyme and monitored the reaction for 15 min at RT (incubations not amenable to a high-throughput platform) for the remaining experiments. The K_m value of fumarate was determined as 41 ± 1.1 µM by monitoring reaction kinetics in a wide range of fumarate concentrations ( Fig. 1C ). Because we sought to identify both activators and inhibitors, the FH assay was performed with a substrate concentration near the K_m value of fumarate (40 µM). A known liability of coupled enzymatic assays is that small molecules appearing to be active from high-throughput screening (HTS) may in fact be modulating the activity of MDH or diaphorase, rather than the target itself. To enable the triage of false positives, we developed an MDH counterassay, omitting FH and adding the MDH substrate malate to initiate the reaction. The kinetics of the MDH counterassay were evaluated at variable concentrations of MDH enzyme and at several time points. The biochemical reaction was found to be in the linear range for the first 5–6 min at concentrations up to 20 IU/mL MDH enzyme ( Fig. 1D ). The K_m value of malate was determined by the MDH assay as 225 ± 30 µM ( Fig. 1E ). This concentration of malate was selected for the MDH counterassay.

To assess the reproducibility and robustness of the assay, the FH assay was performed in white solid-bottom 1536-well Greiner microplates, and statistics were analyzed. Buffer containing enzyme was dispensed into a microplate for columns 1, 2, and 5–48. The reagent without FH enzyme was dispensed into columns 3 and 4 as an enzyme-free negative control. DMSO (20 nL) was transferred into all wells, then incubated with enzyme for 10 min. The reaction was then initiated by adding fumarate. The kinetics of reaction was measured at t = 0 min and t = 15 min ( Table 1 ). As shown in Figure 1F , the signal-to-background (S/B) ratio was 13.8, coefficient of variation was 6.1%, and Z’ factor was 0.83. The assay conditions result in less than 10% substrate conversion during the course of the assay. To ensure the assay is amenable to storage of reagents during the multiple hours that a qHTS assay would require for initiation, enzymatic activity was assessed throughout storage times. FH was found to be unstable when stored at RT, rapidly losing activity within 2 h, but it was quite stable when stored on ice for at least 6 h ( Suppl. Fig. 1 ).

Table 1.

Protocols of Fumarate Hydratase (FH) Assay and Malate Dehydrogenase (MDH) Counterassay.

Protocol of FH Assay.
Step	Parameter	Value	Description
1	Reagent a	3 µL	Dispense reagent a: 13.33 nM fumarate hydratase, 13.33 units/mL MDH, 0.2 mM NAD+, 0.067 mg/mL diaphorase, and 0.067 mM resazurin (in 50 mM Tris pH 8, 5 mM MgCl₂, and 0.01% Brij 35).
2	Reagent b	3 µL	Dispense reagent b: 13.33 units/mL MDH, 0.2 mM NAD+, 0.067 mg/mL diaphorase, and 0.067 mM resazurin (in 50 mM Tris pH 8, 5 mM MgCl₂, and 0.01% Brij 35).
3	Compounds	23 nL	Pin transfer of compounds
4	Time	10 min	Incubate at room temperature
5	Reagent	1 µL	Dispense 1 µL 160 µM fumaric acid
6	Read	525 nm / 598 nm	ViewLux read twice at 15-min intervals
Protocol of MDH Counterassay.
Step	Parameter	Value	Description
1	Reagent a	3 µL	Dispense reagent a: 13.33 units/mL MDH, 0.2 mM NAD+, 0.067 mg/mL diaphorase, and 0.067 mM resazurin (in 50 mM Tris pH 8, 5 mM MgCl₂, and 0.01% Brij 35).
2	Reagent b	3 µL	Dispense reagent b: 0.2 mM NAD+, 0.067 mg/mL diaphorase, and 0.067 mM resazurin (in 50 mM Tris pH 8, 5 mM MgCl₂, and 0.01% Brij 35).
3	Compounds	23 nL	Pin transfer of compounds
4	Time	10 min	Incubate at room temperature
5	Reagent	1 µL	Dispense 1 µL 0.16 mM malic acid
6	Read	525 nm / 598 nm	ViewLux read twice at 5-min intervals

Step notes

Dispense reagent a into the white solid-bottom 1536-well Greiner microplates as follows: rows 1–32 and columns 1–2 and 5–48.

Dispense reagent b into the white solid-bottom 1536-well Greiner microplates as follows: rows 1–32 and columns 3–4.

Compound library was transferred by Pintool.

Microplates were incubated at room temperature for 10 min.

Substrate of FH or MDH was added to the mixture to start the reaction.

Microplates were read in the ViewLux twice, at time 0 min and time 15 min (FH assay)/5 min (MDH assay). Exposure time is 1 s. Excitation/emission is 525 nm/598 nm.

qHTS Screening and Counterscreen

We screened six in-house libraries with a total number of 57,037 small-molecule compounds in 1536-well format ( Fig. 2A ). The libraries include the LOPAC¹²⁸⁰; the NPC, which covers 2816 compounds from late-stage clinical trials and U.S. Food and Drug Administration (FDA)-approved drugs; the NPACT, which contains 5099 annotated compounds with known targets or biological pathways; the MIPE collection with 1912 oncology-focused compounds with well-annotated primary mechanisms of action; a kinase inhibitor–focused library with 977 compounds; and a small-molecule screening collection with diverse scaffolds designed from novel chemical space (44,953 compounds). The assay was robust and consistent throughout the screening plates, with an average assay Z’ factor of 0.73 ± 0.09 ( Fig. 2B ). The hit rate of activators was 0.1% (69 compounds), and of inhibitors was 1.5% (868 compounds). The qHTS data were deposited in PubChem with assay identification (AID) 1347055, and the assay is accessible via the following link: https://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1347055. We cherrypicked 374 compounds based on our selection criteria (active compounds and efficacy ≥50%; see Materials and Methods) for confirmation testing. In the confirmation assay, 13 compounds were confirmed as active activators, and 178 as active inhibitors. The majority of the confirmed hits displayed activity in the MDH counterassay, however, and were triaged.

Figure 2.

Statistics of quantitative high-throughput screen (qHTS) of a human fumarase assay. (A) Flow chart of qHTS of human fumarase assay. In the primary screen, 57,037 compounds from six libraries were screened. Three hundred and seventy-four compounds, which belong to curve classes 1–3 and whose efficacy is more than 50%, were cherrypicked as hits. After a confirmative assay, 166 compounds expanded from four series of inhibitors and two series of activators were picked for follow-up. Twelve compounds from one series of activators were confirmed and selected for the follow-up experiments. (B) Dot plot of the Z factor of a human fumarase assay from 233 primary screen plates. The Z factor ranges from 0.4 to 0.9, and the average is 0.73. (C) Pie charts of hit rates of primary and confirmation screens of human fumarase assays. The compounds were classified as five categories: inactives (curve class 4), active activators (curve classes 1.1, 1.2, 2.1, and 2.2), active inhibitors (curve classes −1.1, −1.2, −2.1, and −2.2), inclusive activators (curve classes 1.3, 1.4, 2.3, 2.4, and 3), and inclusive inhibitors (curve classes −1.3, −1.4, −2.3, −2.4, and −3). The hit rate of the primary screen is shown in the left panel, and the hit rate of the confirmation screen is shown in the right panel.

Only four series of inhibitors and two series of activators survived the counterscreening process. We then plated a total of 166 analogs structurally related to the hits from our in-house library for retesting. One series of activators was confirmed in our FH assay ( Fig. 3A ) and further evaluated in the follow-up experiments. All other compounds dropped out during the reconfirmation assays.

Figure 3.

Heatmap of 166 compounds from six structural series. (A) Hierarchical clustering analysis was done using Spotfire DecisionSite 8.2 (Tibco, Palo Alto, CA). In the heat map, each row represents a compound, and each column represents an assay readout. The left column is the fumarase assay, and the right column is the malate dehydrogenase (MDH) assay. Each cluster of compounds is labeled by the most significantly enriched Medical Subject Headings (MeSH) pharmacological action (PA) term in that cluster measured by a Fisher’s exact test. The colors in the heat map represent the activity of the compound, ranging from blue (inhibitors) to red (activators). (B) Dose–response curve of one series of activators (12 compounds). The x-axis is the logarithmic scale of compound concentration, and the y-axis is the efficacy (%activity) of the compounds. Red curves represent the data from the fumarate hydratase (FH) assay, and gray curves represent the data from the MDH counterassay. The NCATS Chemical Genomics Center (NCGC) number is the sample identification number of each compound in our library.

Identification and Validation of FH Activators

The activator chemotype is composed of a series of phenyl-pyrrolo-pyrimidine-diones, which are active against FH with AC₅₀ ranging from 5.6 µM to 31.6 µM and efficacy ranging from 35% to 79% ( Table 2 ). Compounds were not active in the MDH counterassay ( Fig. 3B ). No signal was detected in the absence of substrates in both assays ( Fig. 4A ), which indicated neither compound was the substrate of those two enzymes (unlike tartrate, vide infra). To ensure activity was not due to impurities in the library material, four compounds (highest potency and efficacy) were purchased from vendors and purified via reverse-phase high-performance liquid chromatography (HPLC). The newly acquired compounds showed similar activity ( Suppl. Fig. 2 ).

Table 2.

One Series of Activators of Fumarate Hydratase.

Sample ID	Library Source	AC₅₀ (µM)	Efficacy (%)	IUPAC Name	Promiscuity Score*
NCGC00102736	Sytravon	8.89	79.36	5-(4-chlorophenyl)-1,3-dimethyl-6-phenethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0804
NCGC00115662	Sytravon	25.06	60.47	6-butyl-5-(4-chlorophenyl)-1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0789
NCGC00115664	Sytravon	17.74	52.03	6-(4-chlorobenzyl)-5-(4-chlorophenyl)-1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0478
NCGC00115666	Sytravon	5.61	35.00	5-(4-chlorophenyl)-1,3-dimethyl-6-pentyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0439
NCGC00115668	Sytravon	15.81	45.48	6-(4-chlorobenzyl)-5-(2-chlorophenyl)-1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0392
NCGC00117671	Sytravon	7.93	60.12	5-(4-bromophenyl)-1,3-dimethyl-6-pentyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0386
NCGC00117675	Sytravon	9.98	78.96	5-(4-bromophenyl)-6-butyl-1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0508
NCGC00117693	Sytravon	7.06	63.74	5-(4-bromophenyl)-1,3-dimethyl-6-phenethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.1055
NCGC00117695	Sytravon	31.55	79.46	5-(4-bromophenyl)-6-(3,4-dimethoxyphenethyl)-1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0488
NCGC00117699	Sytravon	31.55	49.70	5-(2-bromophenyl)-1,3-dimethyl-6-pentyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0293
NCGC00117701	Sytravon	28.12	78.13	6-benzyl-5-(2-bromophenyl)-1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0639
NCGC00117727	Sytravon	25.06	69.96	5-(2-bromophenyl)-1,3-dimethyl-6-phenethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione	0.0425

A promiscuity score is a general assessment of a compound activity profile. It equals the active assays/total assays historically tested in the NCATS database. A compound with a promiscuity score lower than 0.15 is considered a nonfrequent hitter. IUPAC, International Union of Pure and Applied Chemistry.

Table 3.

In Vitro ADME of Top Four Compounds.

Sample ID	Rat Liver Microsome Stability (t1/2, min)	PAMPA Permeability pH 7.4 (1×10⁻⁶ cm/s)	PAMPA Permeability pH 5 (1×10⁻⁶ cm/s)	Solubility (µg/mL)	cLogP
NCGC00102736	1.11	1224	762.26	2.15	3.81
NCGC00117671	6.39	Not conclusive	945.87	<1	4.08
NCGC00117675	7.68	1507	1194	<1	3.57
NCGC00117693	1.23	Not conclusive	590	14.7	3.94

Figure 4.

Evaluation of the series of activators on fumarate hydratase (FH) activity without fumarate and reduction–oxidation (redox) activity in the Amplex assay. (A) No FH activity in the series of activators was shown in the absence of fumarate (substrate of fumarase). The effect of the series of activators on FH or malate dehydrogenase (MDH) activity was evaluated in the FH assay or MDH assay without fumarate or malate, respectively. The protocols were the same except the substrates were omitted from the assays. Red curves represent the data from the FH assay, and gray curves represent the data from the MDH counterassay. (B) No reduction–oxidation reaction (redox) activity of the series of activators was detected in the Amplex Red assay. Redox activity of activators was evaluated in the Amplex Red assay. Two known compounds with different redox activity were used as positive controls. Chloranil has a strong redox activity, and walrycin B has a weak redox activity.

Artefactual liabilities of screening hits can be responsible for the apparent activation, such as the ability to undergo redox cycling. The activator chemotype we identified contains a pyrimidine-2,4(3H)-dione motif. This is not expected to participate in redox chemistry, but it is prudent to de-risk such artefactual activity even if it is not expected. This is especially true when coupled assays such as the one described here use oxidoreductive conversions between NAD+ and NADPH to facilitate assay readout. To examine whether compounds have redox activity, the Amplex Red assay was performed. In the assay, two compounds with weak (walrycin B) and strong (chloranil) redox activity were used as positive controls. None of the hit compounds displayed detectible redox activity ( Fig. 4B ).

We performed MST as a biophysical assay to directly demonstrate binding of activators to FH.²⁴ Two compounds with high potency, NCGC00102736 (AC₅₀ = 8.89 µM) and NCGC00117675 (AC₅₀ = 9.98 µM), were evaluated. The dissociation constant (K_d) of NCGC00102736 was found to be 18.3 µM, and of NCGC00117675 was 23.3 µM ( Fig. 5 ).

Figure 5.

Target engagement of top two compounds with fumarate hydratase (FH). The binding of two compounds with the fumarase enzyme was evaluated by a microscale themophoresis (MST) assay. (Top) MST traces of titrations of NCGC00102736 (A) and NCGC00117675 (B) against 50 nM human FH. The cold region (blue) and hot region (red) were used to determine the Kd of the interaction of compounds and FH enzymes. (Bottom) Dose–response curve for the binding interaction between compounds and FH enzymes. The binding curve yields a Kd of (A) 18.3 µM for NCGC00102736 and (B) 23.3 µM for NCGC00117675.

In Vitro ADME Properties of FH Activators

To evaluate the physicochemical and pharmacokinetic properties of the FH activator series, we selected the four most potent compounds and tested them in a series of in vitro ADME assays. Using automated high-throughput in vitro assays, we examined the rat microsomal metabolic stability, permeability, and solubility in those compounds ( Table 3 ). The half-life (t_1/2) of compounds in the rat hepatic microsomal stability assay ranged from 1.1 min to 7.7 min, suggesting that these compounds were metabolically unstable. The PAMPA was used as an in vitro model of passive, transcellular permeation. Two pH conditions (pH 5 and pH 7.5) were tested in this assay. All compounds scored as highly permeable (>100 × 10⁶ cm/s) at pH 5, and two compounds (NCGC00102736 and NCGC00117675) showed high permeability at pH 7.4. Aqueous solubility was assessed by a high-throughput kinetic solubility assay. The solubility value ranged from <1 µg/mL to 14.7 µg/mL, indicating these compounds had low aqueous solubility.

Evaluation of Prior Art Compounds and Artifacts

Because two inhibitors of FH were reported previously, we evaluated the activity of those inhibitors in our human FH assay. We resynthesized compound 3 (fumarate hydratase-IN-2), which had been reported as active against human FH in a commercial in vitro assay (K_i = 4.5 µM).¹⁴ We found compound 3 to be inactive in our biochemical assay ( Fig. 6A ). Based on the core structure of compound 3, we further cherrypicked 68 analogs of this chemotype and found none of them were active in our FH assay (data not shown). There are multiple reasons for not being able to reproduce the activity of compound 3. For example, the FH inhibition by compound 3 was demonstrated with a commercial biochemical kit, and it is possible that the compound also affected other components of the kit assay system. In another report, Kasbekar¹⁵ reported two inhibitors of tbFH from the same chemotype (compounds 7 and 8 in their article). We tested compound 8, which showed weak inhibitory activity at the highest concentration (IC₅₀ = 42.2 µM; efficacy = 30.2%) in our human FH assay ( Fig. 6A ). Given that enzymes from the two species share identical residues in active sites and overall 59% similarity,²⁵ it is not surprising that compound inhibitors targeting tbFH also showed a weak inhibitory activity against human FH.

Figure 6.

Evaluation of prior art compounds and artifacts. (A) The reported compound 3 and compound 8 were resynthesized and tested in the fumarate hydratase (FH) assay. (B) Activity of a different salt format of the U-50488 compound in the FH assay. (C) Activity of tartrate salt in the FH assay. (D) Time course of the FH assay using tartrate as a substrate for the reaction without adding fumarate. (E) Time course of the malate dehydrogenase (MDH) assay using tartrate as a substrate for the reaction without adding malate. (F) Determine the Km value of tartrate for the MDH enzyme.

During our HTS campaign, we identified an interesting false-positive compound, U-50488, which is a highly selective κ-opioid agonist. There are three salt forms of U-50488 in our collection. We found that the tartrate salt of U-50488 showed very high activator activity. However, the hydrochloride and methanesulfonate salts of U-50488, which are also in our library, showed no activator activity ( Fig. 6B ). We speculated that this activator activity might derive from the tartrate salt component instead of compound U-50488 itself. Potassium sodium tartrate was then tested and was confirmed to produce increased activity in our FH assay ( Fig. 6C ). Because tartrate was reported as a substrate of FH and MDH in the 1960s,^26,27 we hypothesized that tartrate might be used as the substrate of FH or MDH in our assays, giving the appearance of activation. To test this hypothesis, we serially diluted tartrate and added it to FH or MDH assays without their normal substrate (fumarate or malate) in the reaction. We found that tartrate can drive both enzymes in the absence of their eponymous substrates with a similar reaction rate (Fig. 6D and 6E). The K_m value of tartrate was determined by the MDH assay to be K_m = 1296 ± 33 µM ( Fig. 6F ), much higher than malate, the normal substrate of MDH (K_m = 225 ± 30 µM). The finding here is a reminder that the salt composition of compounds can be an interference in an enzymatic assay, which we need to be cautious of in future HTS campaigns. Certainly, this is not an artifact the authors were alert to prior to this study, and the fact that our libraries contained multiple salts of the same compound greatly aided in recognizing the possible cause of the artifact.

Discussion

In our efforts to identify small-molecule probes for FH, we developed a qHTS FH assay and screened a collection of small-molecule libraries. After several rounds of screening and counterscreening, only one series of activators met all activity criteria in our study. Those compounds were not substrates of FH, and they demonstrated direct binding to FH protein. This activator series enhanced the FH activity in the presence of its substrate. Our results suggest that these compounds are allosteric modulators of FH; however, further work is needed to optimize activity and confirm their mechanism of activation.

The series identified as FH activators is a group of phenyl-pyrrolo-pyrimidine-diones (Fig. 2A and Fig. 3B). The hit series consists of 12 active compounds and 9 inactive compounds. A structure–activity relationship (SAR) analysis with this limited set of compounds suggested that the nature of the substitution on the pyrrole nitrogen (R) was important for FH activation ( Suppl. Table 1 ). Active compounds appeared to favor the attachment of a methylene unit to the pyrrole nitrogen, followed by further substitution (see Fig. 2A ). Attachment of additional substituents to this carbon, for example when R was branched such as a phenyl ring or a secondary or tertiary alkyl group, led to erosion in activity. Linear alkyl, phenethyl, and benzyl substituents on the pyrrole N were well tolerated. These data suggest that the pyrrole nitrogen might be important for the compound’s interaction with the protein target and that an uncrowded environment around it might be essential for its binding to the target. With regard to the 5-phenyl ring, the presence of a halide substitution (X = Cl or Br) appeared to be necessary for activity ( Suppl. Table 1 ), because a compound (NCGC00141286) with a long alkyl R but no phenyl ring substitution was inactive. With only one exception, all active compounds in the series have a halogen in the para or ortho position. This information and medicinal chemistry efforts could further improve the chemotype’s potency against fumarase.

Pan-assay interference compounds (PAINS) are the compounds shown as false positives in high-throughput screens.²⁸ To exclude fluorescent compounds, we used change of fluorescence (ΔRFU) as the readout. If compounds are fluorescent, the signal should not change throughout the time (15 min in our assay), and the value of ΔRFU should be close to zero. To filter out the compounds interacting with coupled enzymes in the assay (MDH and diaphorase), a MDH assay was used as a counterassay to triage these false-positive compounds. The MDH counterassay may, however, triage compounds that inhibit or activate both FH and MDH. As a common substrate for both FH and MDH, malate should be able to bind to the active sites of both enzymes, which suggests a structural similarity in active sites between two enzymes. The inhibitors that competitively bind to the same active sites will be filtered out in the MDH counterassay. As such, nonselective competitive inhibitors would be prone to exclusion by assay format. No inhibitors were identified in the current study after screening a collection of 57,037 compounds. In a similar assay format, Kasbekar¹⁵ also didn’t identify any competitive inhibitors for tbFH after screening a collection of 479,984 compounds. Instead, two inhibitors that bind to an allosteric regulatory site were found.

In summary, we developed a quantitative high-throughput screening FH assay and screened a collection of 57,037 small molecules. A series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH, which can serve as a starting point for further optimization and development as probes for the enzyme.

Supplemental Material

DS_873559_Supplementalinformation – Supplemental material for Identification of Activators of Human Fumarate Hydratase by Quantitative High-Throughput Screening

Supplemental material, DS_873559_Supplementalinformation for Identification of Activators of Human Fumarate Hydratase by Quantitative High-Throughput Screening by Hu Zhu, Olivia W. Lee, Pranav Shah, Ajit Jadhav, Xin Xu, Samarjit Patnaik, Min Shen and Matthew D. Hall in SLAS Discovery

Footnotes

Acknowledgements

We thank Monica Kasbekar and Craig J. Thomas for kindly providing the detailed protocol of their fumarase assay; Paul Shinn, Danielle van Leer, and Zina Itkin for their assistance with compound management; Sam Michael, Carleen Klumpp-Thomas, and Jameson Travers for their assistance with automation; Michael Ronzetti and Bolormaa Baljinnyam for technical support of the microscale thermophoresis assay; and Mark Henderson for technical support of the Amplex Red assay.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the intramural research program of the National Center for Advancing Translational Sciences (NCATS).

ORCID iDs

Ajit Jadhav

Matthew D. Hall

Supplemental material is available online with this article.

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