Reagents

CaCl₂ standard was from Thermo Scientific, fatty acid-free BSA from Calbiochem, and atpenin A5 from Santa Cruz Biotechnology. Amplex UltraRed, tetramethylrhodamine methyl ester (TMRM), LysoTracker Red DND-99, and Geltrex were from Invitrogen. Compounds for screening were arbitrarily selected from a diverse, non-combinatorial library obtained from ChemBridge (∼10 mM in DMSO). New stocks of primary hits and their structural analogs were also sourced from ChemBridge (and dissolved to 80 mM in DMSO). The biological activity of iGP-1 (ChemBridge ID 5224148) was validated using compound obtained from Vitas-M (ID STK017597) and found to be essentially identical. All other reagents were from Sigma-Aldrich. sn-Glycerol 3-phosphate was added as disodium rac-α/β-glycerol phosphate (25% active optical isomer sn-glycerol 3-phosphate, 25% inactive optical isomer sn-glycerol 1-phosphate and 50% inactive structural isomer glycerol 2-phosphate) and will be referred to as glycerol phosphate. Where indicated, total and free calcium concentrations in assay buffers were calculated using the Extended MaxChelator program available through Chris Patton on the Stanford University Web site at http://maxchelator.stanford.edu.

Animals

Skeletal muscle mitochondria were isolated from hindlimbs of 5–8 week old female Wistar rats (Harlan Laboratories) as previously described [25]. Cortical synaptosomes were isolated from 6–12 week old male C57BL/6 mice (Jackson Labs) as previously described [26]. The animal protocol was approved by the Buck Institute Animal Care and Use Committee, in accordance with IACUC standards.

Measurement of Mitochondrial H₂O₂ Production and Membrane Potential

Superoxide was measured as H₂O₂ after dismutation by endogenous or exogenous superoxide dismutase. Rates of total superoxide/H₂O₂ production were measured fluorimetrically using Amplex UltraRed in the presence of exogenous superoxide dismutase and horseradish peroxidase in a Varian Cary Eclipse fluorimeter as previously described [27]. Rates of production from specific sites were determined as previously described using the following eight conditions: flavin mononucleotide site of complex I (site I_F) plus matrix NAD-linked dehydrogenases (collectively "site I_F/DH") with 5 mM glutamate, 5 mM malate, and 4 µM rotenone [28]; ubiquinone-binding site of complex I (site I_Q) with either 0.5 or 5 mM succinate [29]; flavin site of complex II (site II_F) with either 15 µM palmitoylcarnitine, 2.5 µM antimycin A, and 2 µM myxothiazol or 0.5 mM succinate, 4 µM rotenone, 2.5 µM antimycin A, and 2 µM myxothiazol [30], [31]; mGPDH with 1.7 mM glycerol phosphate, 4 µM rotenone, 2.5 µM antimycin A, 2 µM myxothiazol, 1 mM malonate, and 250 nM free calcium; outer ubiquinone-binding site of complex III (site III_Qo) with either 0.5 or 5 mM succinate, 4 µM rotenone and 2.5 µM antimycin A [32]. Candidate small-molecule inhibitors were added prior to the start of measurements and H₂O₂ calibration curves were performed in the presence of tested compounds.

Mitochondrial membrane potential (ΔΨm) was estimated fluorimetrically as described previously [33] on the Varian fluorimeter, except the potentiometric dye TMRM was used in quench-mode in place of safranine O [34]. Briefly, skeletal muscle mitochondria (0.2 mg protein ⋅ mL⁻¹) were incubated with 5 µM TMRM in the presence of inhibitors or vehicle. ΔΨm was generated by sequential addition of substrates of complex I (5 mM glutamate plus 5 mM malate), complex II (4 µM rotenone then 0.5 or 5 mM succinate), and mGPDH (2 µM atpenin A5 then 1.7 or 16.7 mM glycerol phosphate). The effect of putative small-molecule inhibitors was normalized as the % change in fluorescence between vehicle treatment and complete depolarization by the uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 2 µM) added at the end of each run. When indicated, the K⁺/H⁺ exchanger nigericin (80 ng ⋅ ml⁻¹) was added to collapse the pH gradient across the mitochondrial inner membrane (ΔpH) and maximize ΔΨm.

The assay buffer for both H₂O₂ and ΔΨm measurements was 120 mM KCl, 5 mM HEPES, 1 mM EGTA, 0.3% (w/v) BSA without or with 250 nM free calcium at pH 7.0. All assays were performed with stirring at 37°C.

Measurement of iGP-1 Fluorescence in Solution

Excitation and emission spectra of iGP-1 fluorescence were acquired with the Varian fluorimeter in standard buffer with pH adjusted to between 1.5 and 11 as indicated.

Screening for Inhibitors of H₂O₂ Production by mGPDH

3,200 compounds were tested initially at 2.5 µM (0.025% DMSO final) in duplicate on different plates against a panel of five distinct assays of site-specific superoxide/H₂O₂ production as described in the previous section with minor changes. Targeted sites included site I_F/DH with 5 mM glutamate, 5 mM malate, and 4 µM rotenone, site I_Q with 5 mM succinate, site II_F with 15 µM palmitoylcarnitine, 2.5 µM antimycin A, and 2 µM myxothiazol, mGPDH with 16.7 mM glycerol phosphate, 4 µM rotenone, 2.5 µM antimycin A, 2 µM myxothiazol, 1 mM malonate, and no added calcium, and site III_Qo with 5 mM succinate, 4 µM rotenone and 2.5 µM antimycin A. Compounds and assay components were distributed by a Biomek FX liquid handling workstation (Beckman) into 96-well black plates (Nunc) to a final volume of 200 µL, and incubated in the dark at room temperature for 30–40 min after the addition of substrate. Endpoint fluorescence was measured on a Perkin Elmer Victor V3 fluorescent plate reader. The fluorescence intensity of each well was normalized as % change from the average of eight DMSO controls on the same plate. Duplicate wells of each of four modulators of mitochondrial metabolism known to inhibit specific sites of superoxide/H₂O₂ production were included on each plate as positive controls and the resulting fluorescence levels set as −100% change from DMSO controls. Known inhibitors included 20 mM aspartate (for site I_F/DH), 1 µM FCCP (for site I_Q), 10 mM malonate (for site II_F), and 2 µM myxothiazol (for site III_Qo). The background fluorescence of the FCCP wells in the site I_Q assay was used to normalize mGPDH plates run in parallel. To minimize position-dependent effects across each assay plate, Tukey’s two-way median polish was applied to fluorescence values after initial normalization [35], [36]. Tukey’s polish is an iterative subtraction of the median row and column values from each well’s given row or column. The procedure is repeated until sequential medians for each row and column are zero and positional gradients in fluorescence are eliminated.

Novel inhibitors of superoxide/H₂O₂ production by mGPDH were identified as compounds whose average effect was to decrease H₂O₂ production by at least 10% without altering H₂O₂ production in other assays by more than ±10%. Select hits in the mGPDH assay were re-sourced from ChemBridge and retested at 0.08–80 µM (0.1% DMSO final) in duplicate on the same microplate as described above against the eight conditions listed under “Measurement of mitochondrial H₂O₂ production and membrane potential.”

Analysis of Structure/activity Relationships

Structure/activity analysis was performed on 18 structural analogs of two structurally-related hits from the primary screen (iGP-1 and iGP-2). These 20 compounds were tested in duplicate at 0.08–80 µM (0.1% DMSO final) in microplate format for effects on site-specific H₂O₂ production as described above. Additionally, four microplate assays for ΔΨm were run in parallel to the H₂O₂ assays. These assays were as described under “Measurement of mitochondrial H₂O₂ production and membrane potential” except that each substrate condition was assayed in a separate microplate. Compounds were tested against the following substrate conditions: 5 mM glutamate and 5 mM malate both without and with 80 ng ⋅ mL⁻¹ nigericin, 5 mM succinate and 4 µM rotenone with 80 ng ⋅ mL⁻¹ nigericin, and 16.7 mM glycerol phosphate and 4 µM rotenone with 80 ng ⋅ mL⁻¹ nigericin. Plates were incubated in the dark at room temperature for 30–40 min (H₂O₂) or 10–15 min (ΔΨm) after the addition of substrate, and endpoint fluorescence was measured on the Victor plate reader. Endpoints were chosen for the H₂O₂ assays to provide good signal-to-noise ratios and linear rates of change without the indicator dye limiting the reaction. Endpoints for the ΔΨm assays were determined in pilot experiments as the times for TMRM accumulation to reach a steady state. At room temperature, this was typically ∼ 5 min and remained stable for at least 30 min. Endpoint fluorescence values were normalized as described in the previous section with wells containing FCCP serving as positive controls for the ΔΨm assays.

cGPDH and mGPDH Activity

The effects of small-molecule inhibitors on cGPDH and mGPDH enzymatic activity were tested according to established procedures. Activity of purified rabbit muscle cGPDH was measured as described by the manufacturer as the rate of oxidation of NADH in the presence of DHAP on the Varian fluorimeter (λ_ex = 365 nm, λ_em = 450 nm). Activity of cGPDH in frozen-thawed synaptosomes was measured at 37°C in a BMG Labtech PHERAstar Plus microplate reader as the linear rate of change in NADH absorbance (λ = 340 nm) specific to the addition of 2.5 mM DHAP and insensitive to 4 µM rotenone. Rates were converted to nmol NADH using an extinction coefficient of 6.22 mM⁻¹ ⋅ cm⁻¹ and a calculated pathlength of 0.6 cm.

Activity of mGPDH in frozen-thawed skeletal muscle mitochondria was measured as 2,6-dichlorophenolindophenol (DCPIP)-linked reduction by glycerol phosphate as described previously in the presence of 1 µM free calcium [27]. Linear rates of change in absorbance (λ = 600 nm) were measured on the PHERAstar microplate reader and converted to rates of DCPIP reduction using an extinction coefficient of 21 mM⁻¹ ⋅ cm⁻¹ and a calculated pathlength of 0.6 cm. Kinetic parameters and inhibitor constants were determined by co-varying glycerol phosphate and novel inhibitors according to recommended procedures [37] and calculated for each data set using GraphPad Prism. Vmax and Km were determined using hyperbolic curves fit to the Michaelis-Menten equation: rate = (Vmax ⋅ [substrate]) ⋅ (K_m+[substrate]) ⁻¹. Hill Slope and IC₅₀ were determined using unconstrained, sigmoidal dose-response curve fits of % remaining activity against concentration of iGP. K_ic and K_iu were determined using plots of rate⁻¹ vs iGP and GP ⋅ rate⁻¹ vs iGP, respectively [37]. Linear regression analysis yielded an average x-coordinate at which the best-fit lines intersect. These x-coordinates correspond to -K_ic and -K_iu in their respective plots [37]. The apparent Vmax of mGPDH is dependent upon the concentration of DCPIP used [13], [38]. Therefore, to determine the maximal activity of mGPDH in isolated synaptosomes, DCPIP was replaced by 1 mM potassium ferricyanide [39] and linear rates of change in absorbance (λ = 420 nm) were converted to rates of ferricyanide reduction using an extinction coefficient of 1.04 mM⁻¹ ⋅ cm⁻¹ and a calculated pathlength of 0.6 cm.

Mitochondrial and Synaptosomal Respiration

The effects of small-molecule inhibitors on respiration were tested in plate-attached skeletal muscle mitochondria using a Seahorse XF24 Analyzer according to published protocols [40]. Briefly, mitochondria (2 or 4 µg protein) were attached to Seahorse assay plates by centrifugation in a mannitol and sucrose-based medium (Seahorse MAS buffer [40]) containing 0.3% (w/v) BSA without or with 250 nM free calcium at pH 7.0. Compounds were titrated to 80 µM on each of four parallel plates with media containing one of the following substrates: 10 mM pyruvate and 0.5 mM malate; 5 mM glutamate and 5 mM malate; 5 mM succinate and 4 µM rotenone; or 16.7 mM glycerol phosphate and 4 µM rotenone with 250 nM free calcium. Compounds were added in these media just prior to loading the assay plate into the Seahorse instrument. Additions were made to set respiration in basal state 2 (substrate only), phosphorylating state 3 (5 mM ADP), non-phosphorylating state 4o (0.5 ug ⋅ mL⁻¹ oligomycin), and to reveal the non-mitochondrial rate (2 µM myxothiazol and 2.5 µM antimycin A). Each concentration of compound was tested in at least two wells on each plate. At least three biological replicates of each titration in each substrate condition were performed.

Synaptosomal respiration was tested in the Seahorse XF24 using published protocols [26], [41]. Synaptosomes (20 or 25 µg protein) were attached to polyethyleneimine and Geltrex-coated Seahorse plates by centrifugation in ionic medium [26], [41] containing 10 mM glucose. Attachment buffer was carefully replaced with ionic buffer containing DMSO, 100 µM iGP-1, 0.5 mM aminooxyacetate, or iGP-1 with aminooxyacetate, and either 10 mM pyruvate or 15 mM glucose. Plates were equilibrated for 20 min at 37°C then loaded into the instrument followed by measurement of basal respiration and injection of 5 µM FCCP (to induce maximal, uncoupled respiration) with 4 µg ⋅ mL⁻¹ oligomycin (to prevent compensatory reversal of ATP synthase). In some experiments, 10 mM oxamate was added with the FCCP/oligomycin to minimize regeneration of cytosolic NAD⁺ by lactate dehydrogenase. Inhibition of the malate-aspartate shuttle was defined as the difference between maximal rates ± aminooxyacetate, expressed as % of control. Inhibition of the glycerol phosphate shuttle was the difference between maximal rates with aminooxyacetate ± iGP-1, as % of control.

Live Cell Imaging

The ability of iGP-1 to cross cellular membranes was evaluated in STHdhQ7 cells cultured at 33°C as described [42]. Cells were seeded on collagen-coated, Lab-Tek chambered coverglass (Nunc) 48 h before use. Prior to imaging, cells were cultured for 45 min with 65 nM LysoTracker Red ±250 nM bafilomycin A1. Medium was then replaced with imaging buffer (120 mM NaCl, 3.5 mM KCl, 0.4 mM KH₂PO₄, 20 mM TES, 5 mM NaHCO₃, 1.2 mM Na₂SO₄, 1.5 mM CaCl₂, 0.5 mM MgCl₂, 2 mM Glucose, and 0.2% BSA, pH 7.4) containing DMSO, 250 nM bafilomycin A1 or 100 µM iGP-1. Cells were imaged on a Nikon Eclipse Ti-PFS inverted microscope equipped with a Cascade 512B camera (Photometrics, Tucson, AZ), a Plan Fluor 100X/1.3 oil objective, an LB-LS17 Xe-arc light source (attenuated), 10–3 excitation and emission filter wheels (Sutter Instruments, Novato, CA), and an MS-2000 linear encoded motorized stage (ASI, Eugene, OR). The filter sets, given as excitation, dichroic mirror, emission in nm/bandwidth, were, for iGP-1 (1 s exposure), 340/26, 409, 460/80 and for LysoTracker Red (100 ms exposure), 543/22, 562, 588/21 (all from Semrock, Rochester, NY). Image acquisition was controlled by NIS Elements 4.0 (Nikon, Melville, NY). Images of cell-free wells containing buffer with DMSO or iGP-1 were used for background subtraction in Image Analyst MKII (Novato, CA).

Data Analysis

Data are presented as mean ± S.E. or mean ±95% C.I. where indicated. Statistical differences between conditions were analyzed by one-way ANOVA with Newman-Keuls post-test as specified in the figure legends. p values <0.05 were considered significant.

Selectivity of Known Inhibitors of mGPDH

Analogs of glycerol phosphate, including glyceraldehyde 3-phosphate and DHAP, are established competitive inhibitors of bacterial, invertebrate, and vertebrate homologs of FAD-linked GPDH [2], [13]–[15]. Because of their limited ability to cross membranes and their involvement in cellular metabolism, these inhibitors are not useful in intact cells. However, neither glyceraldehyde 3-phosphate nor DHAP is oxidized by mitochondria [14] and therefore they may be useful as selective inhibitors of mGPDH in isolated mitochondria. To test this possibility, we measured their effects on the rates of H₂O₂ production and levels of ΔΨm in mitochondria provided with different substrates (Fig. 2).

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Figure 2. Small-metabolite inhibitors of mGPDH are not selective.

(A) Effect of DHAP (black circles) and glyceraldehyde 3-phosphate (white circles) on the rate of H₂O₂ production in the presence of 1.7 mM glycerol phosphate, 4 µM rotenone, 2 µM myxothiazol, 2.5 µM antimycin A, and 250 nM free calcium. The rate of H₂O₂ production by mGPDH was significantly inhibited by each triose phosphate (*p<0.05 versus buffer control; one-way ANOVA with Newman-Keuls post-test). Data are means ± S.E. (n = 3). (B) Effect of glyceraldehyde 3-phosphate on the rate of H₂O₂ production from site I_Q (triangles; 5 mM succinate), site II_F (circles; 0.5 mM succinate, 4 µM rotenone, 2 µM myxothiazol, 2.5 µM antimycin A), and site III_Qo (squares; 5 mM succinate, 3 mM malonate, 4 µM rotenone, 2.5 µM antimycin A). The rate of H₂O₂ production by site II_F was significantly inhibited (*p<0.05 versus buffer control; one-way ANOVA with Newman-Keuls post-test). Data are means ± S.E. (n = 3). (C) Effect of glyceraldehyde 3-phosphate on ΔΨm powered by 5 mM glutamate and 5 mM malate (triangles), 5 mM succinate and 4 µM rotenone (circles), or 16.7 mM glycerol phosphate with 4 µM rotenone and 2 µM atpenin A5 (squares) all in the presence of 80 ng ⋅ ml⁻¹ nigericin to collapse ΔpH. ΔΨm powered by glycerol phosphate was significantly decreased at all concentrations of glyceraldehyde 3-phosphate tested. ΔΨm powered by succinate was significantly increased at 2.5 mM glyceraldehyde 3-phosphate. (*p<0.05 versus buffer control; one-way ANOVA with Newman-Keuls post-test). Data are means ± S.E. (n = 3). GAP, glyceraldehyde 3-phosphate; Glu, glutamate; Mal, malate; Suc, succinate; Rot, rotenone; GP, glycerol phosphate; AtA5, atpenin A5. When not visible, error bars are obscured by the symbol.

https://doi.org/10.1371/journal.pone.0089938.g002

Consistent with their known effects on mGPDH enzymatic activity [2], [13]–[15], both glyceraldehyde 3-phosphate and DHAP inhibited the rate of H₂O₂ production from mGPDH (Fig. 2A). Glyceraldehyde 3-phosphate was more potent, inhibiting nearly 80% at 2.5 mM, but DHAP was less effective. The selectivity of glyceraldehyde 3-phosphate was tested by measuring its effect on H₂O₂ production from site I_Q, site II_F, and site III_Qo. It significantly inhibited the rate of H₂O₂ production from site II_F (Fig. 2B), suggesting a lack of specificity.

As expected, glyceraldehyde 3-phosphate also lowered ΔΨm driven by glycerol phosphate oxidation (Fig. 2C). To test its selectivity in this assay, we also measured ΔΨm driven by glutamate plus malate and by succinate. ΔΨm driven by succinate was significantly increased. This increase in ΔΨm was independent of changes in ΔpH since the K⁺/H⁺ exchanger nigericin was present in all conditions.

We conclude that glyceraldehyde 3-phosphate is not a specific inhibitor of mGPDH but also alters succinate oxidation. Therefore, even in isolated mitochondria, small metabolite inhibitors of mGPDH are limited in utility.

Discovery of Novel Small-molecule Inhibitors of Superoxide/H₂O₂ Production by mGPDH

We recently described a multiple-parallel screen for novel modulators of mitochondrial H₂O₂ production [43]. Each assay in the screen was designed to either drive superoxide/H₂O₂ production predominantly from one mitochondrial redox center or to report undesired effects on ΔΨm (Fig. 3A). Included in this screen was an assay specific for superoxide/H₂O₂ production by mGPDH. This assay was robust with a %CV for intraplate DMSO control wells of 3.4±0.2% (n = 40 plates each tested in duplicate). Out of 3200 compounds arbitrarily selected from a structurally diverse chemical library and tested at 2.5 µM, 87 (2.7%) decreased the signal in the mGPDH assay by at least 10% compared to DMSO controls (Table S1). To identify compounds that were selective for mGPDH, these 87 compounds were counter-screened using a panel of four assays that each targeted a distinct site of superoxide/H₂O₂ production: site I_F/DH, site I_Q, site II_F, and site III_Qo. Compounds that increased or decreased the signal more than 10% in any of these assays were eliminated, leaving seven (0.2%) selective mGPDH inhibitors (Fig. 3B–3G).

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Figure 3. Screening design for novel inhibitors of mitochondrial H₂O₂ production.

(A) Screening workflow. Compounds were screened in duplicate in microplates against six assays. Five assays each targeted a distinct site of superoxide/H₂O₂ production using different substrates without or with inhibitors. The sites assayed were site I_Q, site I_F/DH, site II_F, site III_Qo, and mGPDH. A sixth assay of ΔΨm was used to eliminate general inhibitors of mitochondrial function. All assays were initiated by the addition of Start Solutions containing the substrates and inhibitors listed in parentheses. Details are in “Materials and Methods” and reference 43. Endpoint fluorescence was measured and the effect of compounds was scaled to positive and negative controls included on each assay plate. (B – G) Average normalized effects of 3200 compounds screened against all six assays in duplicate (gray circles). 87 compounds gave >10% inhibition of mGPDH superoxide/H₂O₂ production (dashed line in B). However, after evaluating their effects in the other screens, only 7 were selective for superoxide/H₂O₂ production from mGPDH and did not impair ΔΨm driven with glutamate and malate (red circles in B – G). AUR, resorufin product of Amplex UltraRed oxidation; Suc, succinate; Glu, glutamate; Mal, malate; PalmCarn, palmitoylcarnitine; GP, glycerol 3-phosphate; Rot, rotenone; Ant A, antimycin A; Myx, myxothiazol; Malon, malonate; Nig, nigericin.

https://doi.org/10.1371/journal.pone.0089938.g003

We subsequently obtained four of the most potent and selective of these compounds from the original supplier and retested each over a broad concentration range (0.08–80 µM) against a panel of eight assays of H₂O₂ production targeting the five sites listed above using additional substrate conditions to better evaluate selectivity and potential mechanisms of action. Two compounds were false positives. The other two (iGP-1 and iGP-2; ChemBridge #5224148 and #5224147; Fig. 4) differed structurally by a single atom and each retested well. Another of the seven hits, #5244345, was also structurally similar to these two but displayed less selectivity and was not retested. The most potent compound in the initial screen against H₂O₂ production by mGPDH, iGP-1 (Fig. 4), remained the most potent inhibitor on retesting and also the most selective. The only non-specific effect observed was a weak inhibition of H₂O₂ production from site I_Q at the low concentration of succinate (see below). In contrast, iGP-2 (Fig. 4) was slightly less potent and had stronger off-target effects on sites I_Q and III_Qo. The end result of our unbiased, multiple parallel chemical screens was a set of novel potent inhibitors of superoxide/H₂O₂ production by mGPDH that demonstrated differing selectivity as a result of subtle structural changes. We chose to investigate further the effects of this structural class of compounds on mitochondrial function.

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Figure 4. Structure/activity analysis identifies features conferring mGPDH inhibition.

Summary of effects on H₂O₂ production and ΔΨm of 20 compounds structurally related to the top hit in our primary screen. Each compound was tested against eight assays of site-selective H₂O₂ production and four assays of ΔΨm powered by different mitochondrial substrates (see Materials and Methods for details of individual assays). Data are means of two replicates on the same plate. Five criteria best segregated these compounds according to changes in structural motifs relative to the parent compound iGP-1: potency of inhibition of mGPDH H₂O₂ production (defined by estimating the IC₅₀ concentration or, where an IC₅₀ could not be calculated, the effect on mGPDH H₂O₂ production at 80 µM), effect on ΔΨm powered by glycerol phosphate, effect on ΔΨm powered by glutamate plus malate in the absence or presence of the K⁺/H⁺ exchanger nigericin, and effect on H₂O₂ production by site I_Q driven by succinate. Note that because the method used to normalize mGPDH H₂O₂ production in the screening assay underestimated the background rate, the maximal % inhibition for this assay was ∼70% (see Materials and Methods). Glu, glutamate; Mal, malate; Nig, nigericin; Rot, rotenone; GP, glycerol phosphate; Suc, succinate.

https://doi.org/10.1371/journal.pone.0089938.g004

Analysis of Structure/activity Relationships of Novel mGPDH Inhibitors

Motivated by the observation that subtle changes in structure resulted in changes to both the potency and selectivity of our novel mGPDH inhibitors, we tested an additional 18 compounds structurally related to the top hits in our primary screen to identify structural features that determined the relative potency and selectivity for inhibition of H₂O₂ production by mGPDH. These 20 compounds were retested for effects on eight assays of site-specific H₂O₂ production and four assays of ΔΨm utilizing different mitochondrial substrates (Fig. 4). To identify structure/activity relationships, compounds were placed into four groups according to common generalized structural differences compared to the original parent compound iGP-1 and evaluated for effects on the 12 assays of mitochondrial function to determine shared effects among group members. Several critical conclusions were drawn from this analysis (Fig. 4). First, as was observed in the original round of retesting described above, changing one of the nitrogen atoms in the benzimidazole to oxygen or sulfur (iGP-2 and iGP-3) had little effect on potency against mGPDH yet decreased selectivity. Specifically, these compounds inhibited H₂O₂ production by site I_Q to a greater extent and also increased ΔΨm both in the presence and absence of nigericin. These effects on ΔΨm in the presence of nigericin were subsequently found to be caused largely by artifactual quenching of TMRM fluorescence by the compounds (data not shown). However, the much larger increase in ΔΨm observed in the absence of nigericin was found to represent a true change in ΔΨm, most likely a collapse in the ΔpH component of the protonmotive force. This decrease in ΔpH may explain the greater effect of these structural analogs on H₂O₂ production by site I_Q since this site is known to be uniquely sensitive to ΔpH [44]. Intriguingly, three of four compounds in which additional groups were attached to the free end of the benzimidazole ring were more potent inhibitors of mGPDH (iGP-4– iGP-7). However, these three also had decreased selectivity in similar ways to those observed with changes to the heteroatom of this ring system. The orientation of the benzimidazole and succinamide groups off the central phenyl ring also influenced potency versus mGPDH ROS production. Changing the relative positioning of these groups from ortho- to para- lowered the potency by more than 5-fold (iGP-8). Importantly, altering the carboxyl end of the succinamic acid group decreased potency and selectivity for mGPDH, if any inhibition remained at all (iGP-9– iGP-16). Similarly, the benzimidazole ring was required for inhibition of mGPDH (iGP-17– iGP-20). Ultimately, while our structure/activity analysis yielded no compound with improvements to both potency and selectivity against mGPDH, it provided insight into the structural elements essential to mGPDH inhibition and useful clues as to which chemical features should likely be targeted in future optimization studies.

iGPs Inhibit mGPDH Enzymatic Activity

iGP-1 and iGP-2 did not inhibit ΔΨm driven with glutamate and malate in our initial screen (Fig. 3G). This observation was confirmed during retesting. However, ΔΨm driven by glycerol phosphate was inhibited by several iGPs indicating that these novel compounds inhibited not just H₂O₂ production by mGPDH but also its enzymatic activity (Fig. 4). To investigate this distinction, we assayed the effect of iGP-1 on mGPDH enzymatic activity directly. iGP-1 caused significant inhibition of mGPDH enzymatic activity but, remarkably, did not inhibit the soluble form of GPDH (Fig. 5). Next, we further characterized the effects of the most selective, iGP-1, and the most potent, iGP-5, inhibitors of mGPDH on H₂O₂ production, ΔΨm, and respiration.

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Figure 5. iGP-1 selectively inhibits the mitochondrial isoform of GPDH.

Effect of iGP-1 on the enzymatic activities of mGPDH (black circles) and cGPDH (white circles). iGP-1 significantly inhibited mGPDH activity at all concentrations tested. (*p<0.05 versus vehicle control; one-way ANOVA with Newman-Keuls post-test). Data are means ± S.E. (n = 4 for mGPDH, n = 3 for cGPDH).

https://doi.org/10.1371/journal.pone.0089938.g005

The most selective inhibitor, iGP-1 (Fig. 6A), progressively inhibited H₂O₂ production by mGPDH as its concentration was increased from 0.25 to 80 µM, with a half-maximal effect at about 8 µM (Fig. 6B). Only above 10 µM did iGP-1 start to inhibit H₂O₂ production by site I_Q, demonstrating its good specificity. This effect on H₂O₂ production by mGPDH (Fig. 6B) was mirrored over the same concentration range by significant and specific lowering of ΔΨm driven by glycerol phosphate (Fig. 6C), and significant and specific inhibition of respiratory rates in mitochondria supplied with glycerol phosphate (Fig. 6D), suggesting that iGP-1 inhibited enzymatic activity of mGPDH. iGP-1 decreased H₂O₂ production by site I_Q and ΔΨm driven by low (0.5 mM) succinate but not by high (5 mM) succinate (Fig. 6B, 6C) indicating a subtle off-target effect on succinate oxidation. It had no effect on H₂O₂ production, ΔΨm, or respiration driven by oxidation of a variety of substrates including glutamate, malate, pyruvate, and palmitoylcarnitine.

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Figure 6. iGP-1 selectively inhibits mitochondrial oxidation of glycerol phosphate.

(A) Structure of iGP-1. (B) Effect of iGP-1 on rates of H₂O₂ production from site I_F/DH (black diamonds), site I_Q (with 0.5 or 5 mM succinate; white and black squares, respectively), site III_Qo (with 0.5 or 5 mM succinate; white and black triangles, respectively), mGPDH (black stars), and site II_F (with 0.5 mM succinate or 15 µM palmitoylcarnitine; white and black circles, respectively) measured in a fluorimeter. Details of the conditions to induce H₂O₂ production from each site are given in Materials and Methods. iGP-1 inhibits H₂O₂ production in a site-specific manner from mGPDH. Inhibition of site I_Q H₂O₂ production driven with 0.5 mM succinate alone (white squares) seen at high concentrations of iGP-1 is neither site- nor substrate-selective since site I_Q H₂O₂ production driven with 5 mM succinate (black squares) is not affected and neither site II_F nor site III_Qo H₂O₂ production driven with 0.5 mM succinate (white circles and triangles) is altered. Data are normalized means ± S.E. (n = 4 for mGPDH, n = 3 for all other sites). (C) Effect of iGP-1 on ΔΨm powered by 5 mM glutamate and 5 mM malate without or with 80 ng ⋅ mL⁻¹ nigericin (white or black circles), 4 µM rotenone and 0.5 or 5 mM succinate (white or black triangles), 1.7 mM glycerol phosphate, 4 µM rotenone, and 250 nM free calcium (white squares), or 16.7 mM glycerol phosphate, 4 µM rotenone, and nominal zero free calcium (black squares). ΔΨm powered by glycerol phosphate was significantly decreased in the presence of 8, 25, and 80 µM iGP-1. ΔΨm powered by 0.5 mM succinate was significantly decreased by 80 µM iGP-1 whereas no effect was seen on ΔΨm powered by 5 mM succinate or by glutamate and malate. (*p<0.05 versus vehicle control; one-way ANOVA with Newman-Keuls post-test). Data are normalized means ± S.E. (n = 3–5). (D) Effect of iGP-1 on the rates of mitochondrial respiration driven by 16.7 mM glycerol phosphate with 4 µM rotenone and 250 nM free calcium, 10 mM pyruvate and 0.5 mM malate, 5 mM glutamate and 5 mM malate, or 5 mM succinate and 4 µM rotenone. Respiratory states 2, 3, and 4o were defined by the sequential additions of substrate, 5 mM ADP, and 0.5 µg ⋅ mL⁻¹ oligomycin, respectively. iGP-1 significantly decreased glycerol phosphate-dependent respiration at 25 and 80 µM without altering respiration on other substrates. (*p<0.05 versus vehicle control; one-way ANOVA with Newman-Keuls post-test). Data are means ± S.E. (n = 3 for pyruvate and malate, n = 5 for all other substrates). No significant effects were observed under any condition with iGP-1 at 2.5 and 8 µM (not shown). Glu, glutamate; Mal, malate; Nig, nigericin; Suc, succinate; PC, palmitoylcarnitine; Rot, rotenone; GP, glycerol phosphate; Ca, calcium. When not visible, error bars are obscured by the symbol.

https://doi.org/10.1371/journal.pone.0089938.g006

Similarly, iGP-5 (Fig. 7A) significantly inhibited H₂O₂ production by mGPDH as its concentration was increased from 0.08 to 80 µM, with a half-maximal effect at about 1 µM (Fig. 7B). It also lowered ΔΨm (Fig. 7C) and inhibited respiration (Fig. 7D) driven by glycerol phosphate. iGP-5 lowered ΔΨm driven by glycerol phosphate more potently than iGP-1 (Fig. 6C versus Fig. 7C). However, this structural analog also displayed less selectivity in assays of H₂O₂ production (Fig. 7B) and also significantly increased ΔΨm in the absence but not the presence of nigericin, suggesting a negative effect on ΔpH (Fig. 7C). Further, iGP-5 inhibited respiration driven by pyruvate and perhaps succinate (Fig. 7D).

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Figure 7. iGP-5 inhibits mGPDH activity more potently than iGP-1 but is less selective.

(A) Structure of iGP-5. (B) Effect of iGP-5 on rates of H₂O₂ production from site I_F/DH (black diamonds), site I_Q (with 0.5 or 5 mM succinate; white and black squares, respectively), site III_Qo (with 0.5 or 5 mM succinate; white and black triangles, respectively), mGPDH (black stars), and site II_F (with 15 µM palmitoylcarnitine; black circles) measured in microplate format. Details of the conditions to induce H₂O₂ production from each site are given in Materials and Methods. iGP-5 potently inhibits H₂O₂ production from mGPDH but also causes progressive changes at several other sites of production. Data are normalized means ± ranges (n = 2 technical replicates). (C) Effect of iGP-5 on ΔΨm powered by 5 mM glutamate and 5 mM malate without or with 80 ng ⋅ mL⁻¹ nigericin (white or black circles), 5 mM succinate and 4 µM rotenone (black triangles), or 16.7 mM glycerol phosphate and 4 µM rotenone (black squares). ΔΨm powered by glycerol phosphate was significantly decreased by 2.5 and 25 µM iGP-5. ΔΨm powered by glutamate and malate was significantly increased by 25 µM iGP-5 but only in the absence of nigericin (white circles) suggesting an effect of iGP-5 on the ΔpH component on the proton motive force. (*p<0.05 versus vehicle control; one-way ANOVA with Newman-Keuls post-test). Data are normalized means ± S.E. (n = 3). (D) Effect of iGP-5 on the rates of mitochondrial respiration driven by 16.7 mM glycerol phosphate with 4 µM rotenone and 250 nM free calcium, 10 mM pyruvate and 0.5 mM malate, 5 mM glutamate and 5 mM malate, or 5 mM succinate and 4 µM rotenone. Respiratory states 2, 3, and 4o were defined by the sequential additions of substrate, 5 mM ADP, and 0.5 µg ⋅ mL⁻¹ oligomycin, respectively. iGP-5 significantly decreased glycerol phosphate-dependent state 3 respiration at 25 and 80 µM but also significantly reduced state 3 respiration with pyruvate and malate at 80 µM. (*p<0.05 versus vehicle control; one-way ANOVA with Newman-Keuls post-test). Data are means ± S.E. (n = 3). No significant effects were observed under any condition with iGP-5 at 2.5 and 8 µM (not shown). Glu, glutamate; Mal, malate; Nig, nigericin; Suc, succinate; Rot, rotenone; GP, glycerol phosphate. When not visible, error bars are obscured by the symbol.

https://doi.org/10.1371/journal.pone.0089938.g007

iGP-1 is Cell-permeant

We next determined if iGP-1 was cell-permeant and, therefore, potentially useful for inhibiting mGPDH in situ. Several iGPs exhibited fluorescence with excitation maxima between 320–360 nm and emission maxima between 360–480 nm (Fig. 8A and not shown). Cells treated with 100 µM iGP-1 displayed fluorescence above background, with bright puncta in the cytosol, low levels in the nucleus, and intermediate levels distributed diffusely within the cytosol (Fig. 8B). Timelapse imaging of iGP-1 suggested the punctate fluorescence was in rapidly moving structures independent of the mitochondrial network (not shown). Similar distribution of cellular iGP-1 fluorescence was also observed in several other cultured cell lines (e.g. HEK-293 and PC-3). Co-labeling of cells with iGP-1 and LysoTracker Red DND-99, which localizes to acidic vesicles such as endosomes and lysosomes, revealed a high correlation between the most intense iGP-1 fluorescence and acidic vesicles (Fig. 8B and 8C). Treatment of cells with 250 nM bafilomycin A1, an inhibitor of vesicular H⁺-ATPases, collapsed the vesicular pH gradient (demonstrated by loss of LysoTracker Red staining) (Fig. 8E) and also caused loss of the bright, punctate staining of iGP-1 but not the diffuse fluorescence in the cytosol (Fig. 8D). To address whether iGP-1 was accumulated in acidic vesicles or if iGP-1 fluorescence was enhanced at the lower pH of these vesicles, we measured the fluorescence of iGP-1 as a function of pH. We observed an 8-fold increase in iGP-1 fluorescence as the pH was lowered from pH 7 to pH 1.5 (Fig. 8F). Further, addition of the protonophore FCCP to cells caused a rapid, though incomplete, loss of the bright, punctate staining seen with iGP-1 alone (not shown). Together, these data suggest that the intense punctate staining of iGP-1 is likely the result of a pH-dependent enhancement of iGP-1 fluorescence in acidic compartments and not accumulation in vesicles. Importantly, we can conclude that iGP-1 readily crosses cellular membranes and therefore should have access to mGPDH in intact systems.

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Figure 8. iGP-1 is cell-permeant and inhibits mGPDH in intact presynaptic terminals.

(A) Excitation and emission spectra of 0.4 mM iGP-1 with peaks at 342 nm and 378 nm, respectively. (B) Fluorescence of 100 µM iGP-1 in a live STHdhQ7 cell labeled with LysoTracker Red. (C) Fluorescent labeling of acidic vesicles by LysoTracker Red in the same cell shown in (B). Insets in (B and C) highlight colocalization between intense iGP-1 fluorescence and LysoTracker Red labeled vesicles. (D) Fluorescence of 100 µM iGP-1 in a live STHdhQ7 cell pretreated with 250 nM bafilomycin A1. (E) LysoTracker Red fluorescence in the same cell pretreated with bafilomycin A1 as in (D). Scale bar in (B – E) = 10 µm. (F) iGP-1 fluorescence as a function of buffer pH (ex. λ = 342 nm, em. λ = 430 nm). (G) Effect of vehicle control (DMSO, white bars), 100 µM iGP-1 (light gray bars), 0.5 mM aminooxyacetate (dark gray bars), or their combination (black bars) on synaptosomal respiration. Basal respiration was measured in the presence of either 10 mM pyruvate or 15 mM glucose followed by the addition of 5 µM FCCP and 4 µg ⋅ mL⁻¹ oligomycin to induce uncoupled respiration without or with (rightmost condition) 10 mM oxamate. Oxamate was included to minimize regeneration of cytosolic NAD⁺ by lactate dehydrogenase. Respiration on pyruvate alone was not altered by inhibition of either or both of the NADH shuttle systems. Both basal and uncoupled respiration on glucose was unaffected by iGP-1 alone but significantly decreased by aminooxyacetate alone. Oxamate increased the maximal rate of respiration achieved by glucose alone. In the presence of oxamate, the combination of iGP-1 combined with aminooxyacetate consistently decreased the maximal uncoupled rate compared to aminooxyacetate alone. (*p<0.05 versus vehicle control and iGP-1 alone; one-way ANOVA with Newman-Keuls post-test). Data are means ± S.E. (n = 3 for pyruvate, n = 4 for glucose, n = 5 for glucose ± oxamate). (H) Assignment of the % of maximal respiration that is dependent on the NADH shuttles in the presence of glucose and oxamate. Under this condition of high glycolytic demand, there is a significant dependence upon both the malate-aspartate shuttle and the glycerol phosphate shuttle. (*p<0.05 versus no change from vehicle control; ^#p<0.05 versus no change from aminooxyacetate). Data are means ±95% C.I. (n = 5). AOA, aminooxyacetate; Pyr, pyruvate; F/O, FCCP with oligomycin; Glu, glucose.

https://doi.org/10.1371/journal.pone.0089938.g008

Effect of iGP-1 in Cellular Systems

mGPDH is a critical component of the glycerol phosphate shuttle for regenerating cytosolic NAD⁺. However, because of a lack of cell-permeant selective inhibitors of mGPDH and compensation by other NAD⁺ regenerating mechanisms, there is a lack of evidence of functional mGPDH activity in intact systems. We tested the role of mGPDH-dependent metabolism using iGP-1 in synaptosomes because mGPDH is active in neurons [27], [45], [46], there is a significant role for NADH shuttles in synaptic energetics [26], [47], there are no reports on neural function in mGPDH^−/− mice, and synaptosomes are a tractable intact system for studying neuronal energetics under tightly controlled cell-, substrate-, and demand-specific conditions.

We verified that the high mGPDH activity reported in brain was present in synaptosomes. In frozen-thawed cortical presynaptic terminals, mGPDH activity was high (207±16 nmol ferricyanide reduced ⋅ min⁻¹ ⋅ mg protein⁻¹; mean ± SE, n = 3). To demonstrate the likelihood of a functional shuttle system, we measured cGPDH activity in the same preparations. There was a significant rate of DHAP-specific, rotenone-insensitive NADH oxidation that we attribute to cGPDH (22±1 nmol NADH oxidized ⋅ min⁻¹ ⋅ mg protein⁻¹; mean ± SE, n = 3).

We then applied iGP-1 alone or in combination with aminooxyacetate (to inhibit the malate-aspartate shuttle) (Fig. 1B) in synaptosomes respiring on glucose (Fig. 8G). Maximal demand was created by adding FCCP (plus oligomycin to decrease ATP depletion). In control experiments, pyruvate replaced glucose to bypass the need for NAD⁺ regeneration (Fig. 1). 100 µM iGP-1, 0.5 mM aminooxyacetate, or their combination had no effect on basal or maximal respiration with pyruvate (two left hand sets of columns, Fig. 8G). With glucose as substrate, basal and maximal respiration were inhibited by aminooxyacetate (two middle sets of columns, Fig. 8G), confirming a role for the malate-aspartate shuttle in synaptic bioenergetics [26], [47]. However, iGP-1 had no effect even in combination with aminooxyacetate, suggesting little contribution of the glycerol phosphate shuttle.

In the absence of inhibitors, glucose supported slower respiration than pyruvate. As the result with aminooxyacetate demonstrates, NAD⁺ regeneration has a significant permissive role in synaptosomal metabolism of glucose but not pyruvate. There was also faster extrasynaptosomal acidification with glucose as substrate, suggesting efflux of lactate (with H⁺) to regenerate cytosolic NAD⁺ at lactate dehydrogenase (Fig. 1C). This would divert pyruvate destined for respiration and cause slower respiration with glucose than with pyruvate. To test this hypothesis, we added oxamate (with the FCCP and oligomycin) to inhibit lactate dehydrogenase during oxidation of glucose (Fig. 1C). This should maximally drive respiration while also placing the greatest demand on the NADH shuttles to facilitate glycolysis. Maximal respiration was substantially faster in the presence of oxamate (two right hand sets of columns, Fig. 8G), and aminooxyacetate still inhibited significantly. Importantly, although iGP-1 alone had no effect, it caused consistent additional inhibition in the presence of aminooxyacetate. Thus, there was a significant dependence of uncoupled respiration on both the malate-aspartate shuttle (45.9±0.7%) and the glycerol phosphate shuttle (5.3±0.9%) (means ± SE, n = 5) (Fig. 8H).

Characterization of the Effects of Novel Inhibitors on the Kinetics of mGPDH

The greater potency of iGP-5 versus iGP-1 was confirmed against both mGPDH activity (Fig. 9A and Table 1; IC₅₀ = 1.0 µM and 6.3 µM, respectively) and H₂O₂ production (Fig. 9B and Table 1; IC₅₀ = 1.0 µM and 13.6 µM, respectively). Inhibition of mGPDH by both iGP-1 and iGP-5 displayed a selective, one-site binding profile (Table 1; Hill slopes 0.92 and 0.85, respectively). These data support the conclusion that these novel compounds inhibit mGPDH enzymatic activity and confirm that iGP-1 is the most selective of the potent inhibitors we have identified. We next characterized the mechanism of action of this novel class of mGPDH enzymatic inhibitors by measuring the effects on the kinetics of mGPDH. We assayed mGPDH activity at different concentrations of glycerol phosphate and inhibitor (either iGP-1 or iGP-5). These data (Fig. 10A, 10B) showed that the maximum observed rate of DCPIP reduction by mGPDH decreased as the inhibitor concentration increased.

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Figure 9. iGP-1 and iGP-5 potently inhibit mGPDH enzymatic activity and H₂O₂ production.

(A) iGP-5 (black circles) is a more potent inhibitor of mGPDH enzymatic activity than iGP-1 (white circles). IC₅₀ concentrations were 1.0 and 6.0 µM for iGP-5 and iGP-1, respectively. Data are means ± S.E. (error bars) (n = 3–5). (B) iGP-5 (black circles) is a more potent inhibitor of mGPDH H₂O₂ production than iGP-1 (white circles). IC₅₀ concentrations were 1.0 and 14.2 µM for iGP-5 and iGP-1, respectively. Data are means ± S.E. (error bars) (n = 2–4). When not visible, error bars are obscured by the symbol.

https://doi.org/10.1371/journal.pone.0089938.g009

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Figure 10. iGP-1 and iGP-5 produce mixed inhibition kinetics.

(A) Effect of co-varying iGP-1 and glycerol phosphate on mGPDH activity. Curves are hyperbolic best-fits of the Michaelis-Menten equation: mGPDH activity = (Vmax ⋅ [Glycerol phosphate]) ⋅ (K_m+[Glycerol phosphate])⁻¹. Data are means ± S.E. (n = 3–4). (B) Effect of co-varying iGP-5 and glycerol phosphate on mGPDH activity. Data are means ± S.E. (n = 3–5). (C) Double reciprocal plot of the data in (D). Data are means ± S.E. (n = 3–4). (D) Double reciprocal plot of the data in (E). Data are means ± S.E. (n = 3–5). (E) Effect of iGP-1 (white circles) and iGP-5 (black circles) on the V_max of mGPDH activity. V_max values at each concentration of compound were calculated for each experiment using best-fit Michaelis-Menten curves similar to those in (D and E). The dashed line denotes the mean value for the vehicle control. Data are means ± S.E. (error bars) (n = 3–5). (F) Effect of iGP-1 (white circles) and iGP-5 (black circles) on the K_m of mGPDH. K_m values at each concentration of compound were calculated for each experiment using best-fit Michaelis-Menten curves similar to those in (D and E). The dashed line denotes the mean value for the vehicle control. Data are means ± S.E. (n = 3–5). (G) Scheme for an enzymatic reaction in which enzyme E converts substrate S into product P in the presence of an inhibitor I that displays both competitive and uncompetitive behavior defined by inhibitor dissociation constants K_ic and K_iu, respectively [43].

https://doi.org/10.1371/journal.pone.0089938.g010

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Table 1. Inhibition parameters for iGP-1 and iGP-5.

https://doi.org/10.1371/journal.pone.0089938.t001

Analysis of double reciprocal plots (Fig. 10C, 10D) showed that each inhibitor lowered the V_max and increased the K_m for glycerol phosphate. Fig. 10E shows that the apparent V_max was progressively decreased by each inhibitor with iGP-5 more potent than iGP-1. Fig. 10F shows that the K_m for glycerol phosphate was progressively increased; again, iGP-5 was more potent than iGP-1. This profile of lowered V_max combined with a change in K_m is indicative of a mixed inhibitor (Fig. 10G) that interacts competitively with respect to the substrate (defined by the competitive inhibitor dissociation constant K_ic) and uncompetitively with the enzyme-substrate complex (defined by the uncompetitive inhibitor dissociation constant K_iu). Values for these dissociation constants were in the 10 µM range for iGP-1 and the 1 µM range for iGP-5 (Table 1).