Innovative electrochemical screening allows to identify new

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Innovative electrochemical screening allows to identify new transketolase inhibitors Chloe M.G. Aymard, Matilte Halma, Arnaud Comte, Christine Mousty, Vanessa Prevot, Laurence Hecquet, Franck Charmantray, Loïc Jacques Blum, and Bastien Doumèche Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01752 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 28, 2018

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Analytical Chemistry

Innovative electrochemical screening allows to identify new transketotransketolase inhibitors Chloé M.G. Aymard[a], Matilte Halma[b], Arnaud Comte[a], Christine Mousty[b], Vanessa Prévot[b], Laurence Hecquet[b], Franck Charmantray*[b], Loïc J. Blum[a] and Bastien Doumèche*[a] Phone: +334 72 43 14 84 E-mail: [email protected] [a]

Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, ICBMS UMR 5246 CNRS, Université de Lyon, Université Lyon 1, CNRS, INSA Lyon, CPE Lyon. 43 bd du 11 Novembre 1918, 69622 Villeurbanne Cedex (France). [b]

Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, ICCF UMR 6296 CNRSUCA-Sigma F-63000 Clermont-Ferrand (France). ABSTRACT: Transketolases (TK) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the non-oxidative branch of pentose phosphate pathway. They are considered as interesting therapeutic target in numerous diseases and infections (e.g. cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, enzyme loading, electrochemical protocol and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 minutes that allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identify to inhibit TK from E. coli with an IC50 of 63 µM and an inhibition constant (KI) of 3.4 µM. The electrochemical assay was also used to propose an inhibition mechanism.

KEYWORDS: Transketolase, Electrochemistry, High Throughput Screening, Inhibitors.

Introduction Transketolase (TK, EC 2.2.1.1) is a key enzyme of the nonoxidative branch of the pentose phosphate pathway (PPP). TK catalyzes the transfer of a two carbon unit from a ketose phosphate (donor substrate) to an aldose phosphate (acceptor substrate), in the presence of thiamine pyrophosphate (TPP) and magnesium as cofactors.1 This enzyme follows a Bi Bi Ping Pong mechanism (Scheme 1, way a): after TPP deprotonation (leading to the active form of TPP), this latter attacks the donor substrate leading to the release of the aldehyde product along with the formation of the carbanion intermediate, α,βdihydroxyethylthiamine diphosphate (DHETPP).2,3 This reactive intermediate transfers the two carbon unit to the aldehyde acceptor substrate through C-C bond formation. The ketone product is then released and the TPP is regenerated. TK is an ubiquitous enzyme implicated in the production of D-ribose-5-phosphate required for the synthesis of nucleic acid4 and in the production of Derythrose-4-phosphate necessary for the biosynthesis of aromatic amino-acids (shikimate pathway in plants and microorganisms).5 PPP is also the major biochemical pathway that generates antioxidant NADPH.6 Previous

works have shown that the PPP can be over-activated in the context of abnormal proliferation of cancer cells and TK is overexpressed in this pathological situation.7–9 This up-regulation allows to meet the increased demand of ribose-5-phosphate for nucleotide synthesis7 and the demand of NADPH to counteract the production of reactive oxygen species enabling cancer cells to survive.6 Moreover, this enzyme may be deficient in the context of neurodegenerative diseases such as Alzheimer's disease10,11, Beriberi and Wernicke-Korsakoff syndrome.12–14 Furthermore, recent studies have described the importance of TK on the survival of Mycobacterium tuberculosis, the aetiologic agent of tuberculosis.15 Finally, TK from Plasmodium falciparum also appears to be a drug target for the treatment of malaria.16 In this context, TK appears to be an interesting therapeutic target for which it is important to find specific and efficient inhibitors, relevant to the disease. Several molecules have been reported to inhibit TK from different sources with different inhibition constants (KI). Oxythiamine (KI = 1.4 mM)17, thiamine (KI = 34 mM)18, pyrophosphate (KI = 0.28 mM)18, analogs of the cofactor TPP, and hydroxyphenylpyruvate (KI = 3 mM)19,

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an analog of the donor substrate, inhibits TK from Saccharomyces Cerevisiae. D-arabinose-5-phosphate inhibits TK from Escherichia Coli with a KI of 6 mM.20 More recently, an in-silico screening of human TK inhibitors based on diphenyl urea derivatives was performed and the best candidates led to IC50 comprised between 0.1 and 0.2 mM.21 Finally, a docking study was performed with the TK from Plasmodium falciparum leading to the synthesis of 4-anilinoquinoline triazines derivatives with IC50 comprised between 0.072 and 0.085 mM.22 However, these values are generally considered as too high for further pharmaceutical development for which early lead compounds should exhibit IC50 below 0.1 mM.

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phenol oxidase32 or galactose oxidase33 as secondary enzyme. To avoid the use of auxiliary enzymes, we have reported on recently the monitoring of TK activity by following the oxidative trapping of TK intermediate DHETPP.34 This intermediate can be oxidized by a redox mediator such as porphyrindin, ferrocytochrome or phenazine methosulfate but ferricyanide (Fe(CN)63-) remains the most economic and available oxidant.35 The application of a potential of +0.5 V vs. Ag/AgCl allows the re-oxidation of this mediator. This last method takes the advantage to proceed without coupled enzyme and using only the donor substrate as the unique TK substrate (Scheme 1, way b).

Scheme 1: Reaction mechanism of TK in the presence of TPP, donor and acceptor substrates (way a). Principle of the electrochemical detection of TK activity (way b): the reaction intermediate DHETPP is oxidized by ferricyanide. In order to find some new efficient TK inhibitors, a rapid and sensitive enzymatic assay is a prerequisite. Initially, TK activity can be measured either by spectrophotometry by using NADH-dependent auxiliary enzymes23,24, or chromophores such as phenol red used in a pH-based high throughput assay25 or as tetrazolium red which oxidizes non-α hydroxylated substrates26 or by fluorescence using modified substrates.27 However, these optical methods require a secondary enzyme, are limited by a lack of sensitivity and specificity or by the harsh synthesis of unnatural fluorescent substrates. Another spectrophotometric method based on the reduction of potassium ferricyanide by the DHETPP intermediate was also described28. This assay displays low sensitivity due to the low molar absorption coefficient of reduced ferricyanide (εM ≈ 1000 M-1.cm-1). Moreover, it requires large amount of TK enzyme. TK activity can also be assayed by HPLC or GC analysis29–31, nevertheless, these methods require sophisticated and expensive instrumentation and cannot be easily adapted to a screening protocol because they are highly time consuming. On the other hand, electrochemical methods allowed to measure TK activity using poly-

Scheme 2: Exploded scheme of the electrochemical plate (a): the printed circuit board (PCB) is overlaid by the 96 screen-printed carbon working electrodes and Ag/AgCl counter/reference electrodes, covered by a perforated plastic foil. Principle of TK activity detection in the presence of L-erythrulose (L-ery) as donor substrate (b): DHETPP is oxidized by Fe(CN)63leading to glycolic acid and Fe(CN)64- re-oxidized at the electrode surface. Main analytical performances of published colorimetric, fluorescence-based and electrochemical methods for


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TK activity measurement are summarized in Supporting information table S1. However, these amperometric methods are also timeconsuming because only few samples can be measured at a time and large reaction volumes are often required. To overcome these limitations, we previously proposed an approach to detect 96 enzymatic activities within a short timeframe (1 to 3 min) in a small reaction volume (30-50 µl) using intermittent pulse amperometry (IPA).36–38 Briefly, 96 electrodes are screen-printed onto a printed circuit board acting as connector with special potentiostat (Scheme 2a). The 96 electrodes are delimited by a perforated plastic foil to receive the reaction media. This configuration allows a rapid optimization of electrochemical parameters and reactional conditions. This device was used to screen the activities of dehydrogenases and laccases from enzyme libraries by measuring electron transfer between the enzymes and the electrodes36,37 or to detect hydrogen peroxide produced by oxidases using amperometry or electrochemiluminescence.38 Here we applied this system to detect TK activity based on the Fe(CN)6 mediated oxidation of DHETPP (Scheme 2b). Optimization of this enzymatic assay has been performed with the easily available TK from E. Coli, as a proof of concept. After the characterization of electrodes and optimization of the activity assay, the 96-well format electrodes have been used to screen more than 1,300 molecules of an in-house chemical library in order to find new inhibiting early lead compounds. Finally, kinetic studies were performed to identify the mechanism of inhibition.

Experimental section 1.

Reagents

Bis(triphenylphosphine)palladium(II) dichloride (PdCl2(PPh3)2), bovin serum albumin (BSA), bromopyruvic acid, cesium carbonate (Cs2SO3), dichloromethane (CH2Cl2), dimethylsufoxide (DMSO), 1,4-dioxane, L-(+)erythrulose (L-ery), ethyl acetate, glycolaldehyde dimer (GA), glycyl-glycine (gly-gly), 4-hydroxyphenylpyruvic acid (HPP), lithium β-hydroxypyruvate hydrate (HPA), 4(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes), magnesium sulfate monohydrate (MgSO4.H2O), reduced nicotinamide adenine dinucleotide (NADH), oxythiamine chloride hydrochloride, petroleum ether, potassium ferrocyanide trihydrate (K4Fe(CN)6.3H2O), potassium pyrophosphate (PPi), D-ribose-5-phosphate (R5P), sodium bicarbonate (NaHCO3), tetrahydrofuran, thiamine hydrochloride, thiamine pyrophosphate (TPP), triethylamine, tris(dibenzylideneacetone)dipalladium(0) (Pd2dba3), 4,5-Bis(diphenylphosphino)-9,9dimethylxanthene (XantPhos) and yeast alcohol dehydrogenase (25 µmol.min-1.mg−1) were purchased from Sigma– Aldrich (Saint-Quentin-Fallavier, France). 2-aminopyrazine was purchased from Acros Organics (Geel, Belgium) and 1-ethoxy-4-ethynylbenzene from TCI (Zwijndrecht, Belgium). Potassium ferricyanide (K3Fe(CN)6) and magnesium chloride (MgCl2) were pur-

chased from Prolabo (Nantes, France) and potassium chloride (KCl) was obtained from BDH (Poole, England). TK was produced as previously describe by Touisni et al.34 (Supporting information) Nanoparticles of Mg2Al-NO3 layered double hydroxides (LDH) were synthetized at ICCF as previously described by Halma et al.33 2.

Electrochemical System

Ninety-six electrodes were screen printed on a printed circuit board (PCB) (Cirly SA, Brignais, France). The diameter of the working electrode was 3 mm. The twoelectrode system was composed of a working electrode made of carbon (Electrodag 6038SS) and a counter/reference electrode in Ag/AgCl (Electrodag PF-407C) (Acheson, Scheemda, Netherlands) printed by using a DEK 248 device (Weymouth, United Kingdom). The inks were printed and cured in this order: Ag/AgCl followed by carbon. Ag/AgCl ink was cured at 120°C and carbon at 80°C for 15 min. Electrochemical assays were performed with an AndCare 9600 sensor (Alderon Biosciences Inc., Beaufort, USA), which allows to apply a potential on the 96 electrodes at the same time. Measurements were performed by intermittent pulse amperometry (IPA): series of millisecond pulses of potential were applied to the working electrode, separated by periods during which the circuit was open. Electrodes were used once before recycling of the printed circuit board by washing with acetone as described previously.36–38 3.

Electrochemical TK activity measurement

Characterization of the electrodes was performed by cyclic voltammetry by using a multipotentiostat VMP3 (Bio-logic, Claix, France). Due to the screen-printing method, the electrode surface slightly varied from one well to the next. To take these variations into account, carbon electrodes were first pretreated by oxidation in the presence of 1 M carbonate buffer39 using IPA and by applying a potential of +1.2 V vs. Ag/AgCl for 10 min, with pulses of 82 ms and a frequency of 1 Hz.38 All enzymatic reactions were measured in 30 µL of 0.05 M Hepes buffer pH 7.0 containing 0.1 M KCl as additional electrolyte. The reaction media was composed of 0.1 mM K3Fe(CN)6, 1 mM MgCl2, 13.9 µg of TK and TPP (0-200 µM). The enzymatic reaction was started by the addition of 5 µL of 0-7 mM L-ery and the response was measured after 5 min of reaction. The ferrocyanide (Fe(CN)64-) produced by the oxidation trapping of DHETPP was oxidized by IPA with a potential of +0.56 V vs. Ag/AgCl for 2 min, with pulses of 82 ms and a frequency of 1 Hz. The ten last measurements were averaged and used as a representative current intensity on the electrode. Current intensities were corrected by values obtained before the addition of L-ery. All measurements were done at least 3 times (n ⩾ 3). Each response curve was fitted using a single rectangular hyperbola model exhibiting the equation (1): J × [S] J = () K

+ [S] Where J is the current density (µA.cm-2), Jmax the maximal current density obtained at substrate saturation, [S]



the concentration of L-ery or TPP and KM app the apparent Michaelis-Menten constant. These conditions were optimized to afford less than 10% of conversion of L-ery. Electrochemical response was measured in the presence of 13.9 µg TK in solution or previously adsorbed alone at the screen printed electrode surface or co-absorbed with the same amount of layered double hydroxides (TK@LDH 1:1) as previously described.34 Then, electrochemical response was measured in the presence of known TK inhibitors selected from the literature. The reaction media was supplemented with 1 mM of inhibitor (thiamine, oxythiamine, pyrophosphate or hydroxyphenylpyruvate) and signals were expressed as a percentage of inhibition compared to control without inhibitor. Each inhibitor was assayed 4 times (n=4). 4.

Determination of screening assay quality using Zfactor

Z-factor was determined with 40 electrodes in the presence of 0.1 mM potassium ferrocyanide (Fe(CN)64-) or using the reaction media described in the previous paragraph (with 0.01 or 0.2 mM TPP and 1 mM L-ery). Z-factor was calculated using the equation (2)40: 3 SD (control) + 3 SD (sample) Z − score = 1 − ($) mean of sample − mean of control

Where SD is the standard deviation, control is the signal obtained with only Hepes buffer or with the reaction media without TPP and sample is the signal obtained with 0.1 mM ferrocyanide or after 5 min of enzymatic reaction in the presence of TPP (0.01 or 0.2 mM). A Z-factor comprised between 0.5 and 1 refers to an excellent screening assay. If it is comprised between 0 and 0.5, an intern control is required to validate signals. If it is equal to zero, this screening is a “yes/no” type assay and if it is inferior to zero, the screening is essentially impossible. 5.

Screening of ICBMS’ chemical library

A total of 1360 from the chemical library of ICBMS were screened at a final concentration of 0.1 mM. Samples were dissolved in DMSO corresponding to a final concentration of 10% (v/v) in the electrochemical assay. 4 electrodes were used as positive control in the presence of 0.01 mM TPP and 4 electrodes as a negative control without TPP. The 88 other electrodes allow to screen the library. Each molecule was assayed 4 times (n=4). Signals were averaged and expressed as percentage of inhibition compared to the positive control. Compounds displaying more than 50% of inhibition were selected as hits. 6.

Synthesis of I38-49

The best inhibitor obtained from the screening, 2-(4ethoxyphenyl)-1-(pyrimidin-2-yl)-1H-pyrrolo[2,3b]pyridine, was synthetized as previously described by Dimanche-Boitrel et al.41, following a 2-step synthesis (Supporting information). 7.

Determination of enzymatic mechanism by IPA

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(0-100 µM) by IPA using electrochemical parameters described above. The discrimination mode of Dynafit software was used to determine the most probable inhibition mechanism of I38-49.42 Seven mechanisms were compared (Supporting information S8): competitive, uncompetitive, non-competitive, mixed non-competitive, partial uncompetitive, partial non-competitive and partial mixed non-competitive. The Akaike information criterion (AIC), an estimator of the quality of a model relative to others, is used to discriminate mechanisms as it helps to limit the overparameterization. Inhibition constants (KI) and apparent Michaelis Menten constants (KM app) were determined according to this mechanism. Results and discussion 1.

Electrochemical detection of ferri/ferrocyanide with IPA

The screening assay will rely on the quality of our home-made screen-printed electrodes. This quality was first controlled by cyclic voltammetry. The detection of ferrocyanide (Fe(CN)64-) was characterized following the Laviron’s theory.43 Cyclic voltammetry was performed between -1 and +1 V vs. Ag/AgCl with different scan rates ranging from 0.005 to 4 V.s-1 in the presence of 1 mM ferricyanide/ferrocyanide (Fe(CN)63-/ Fe(CN)64-) in 0.05 M Hepes buffer pH 7.0, 0.1 M KCl. A linear dependency between the oxidation or reduction peak currents and the square root of the scan rate is observed (Supporting information S1a), which shows that the oxidation and reduction of Fe(CN)63-/ Fe(CN)64- is controlled by diffusion, as expected for soluble mediator. The plot of the oxidation and reduction potentials versus the logarithm of the scan rate (Supporting information S1b) shows a linear dependency. The heterogeneous apparent surface electron-transfer rate constant (12.9 s-1), the electron-transfer coefficient (0.4) and the diffusion coefficient of Fe(CN)63-/ Fe(CN)64- to the electrode (6.1 x 10-6 cm².s-1) are comparable to those obtained with other carbon paste electrodes.44 This characterization of the carbon electrodes allowed us to conclude that the quality of the screen printing is sufficient to correctly detect Fe(CN)64by intermittent pulse amperometry (IPA). As described before38, all the electrochemical parameters (potential, pulse width and frequency of pulses) were optimized for the detection of Fe(CN)64- to achieve the following optimal conditions: a potential of +0.56 V vs. Ag/AgCl was applied during two minutes with pulses of 82 ms and a frequency of 1 Hz. Calibration curve of 0.0510 mM Fe(CN)64- was obtained using 16 independent electrodes (and 16 different Fe(CN)64- concentrations). This calibration curve shows a linear range between 0.1 and 6 mM with a sensitivity of 212 µA.mM-1.cm-2 (Supporting information S2). The limit of detection (LOD) is 0.05 mM, which is the lowest concentration assayed that exhibits a signal/noise (S/N) ratio greater than 3. 2.

TK activity measurements by IPA

Electrochemical response was measured in the presence of different concentrations of TPP (0-200 µM) and I38-49


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After having determined the characteristics of the electrodes, this assay was used to measure TK activity by oxidation of the TK intermediate DHETPP thanks to Fe(CN)63- oxidation. This mediator is reduced in Fe(CN)64and the application of a potential of +0.56 V vs. Ag/AgCl by IPA allows its oxidation at the electrode surface (Scheme 1 way b).

ble 1). With high concentration of TPP (>40 µM), more than 10% of substrate is converted into products, suggesting that the signal is not limited by the enzymatic reaction but by the chemical step (i.e. the oxidation of DHETPP by ferricyanate). Moreover, others donor substrates (D-fructose-6-phosphate and hydroxypyruvic acid) were also assayed. Despite of lower KM app for hydroxypyruvic acid and D-fructose-6-phosphate (43 µM and 100 µM respectively, against 156 µM for L-ery), performances of our electrochemical assay (sensitivity, Jmax and linear range) are optimal when L-ery is used as donor substrate. Moreover, L-ery is an inexpensive and commercially available product and has already been described as a ketone donor in place of natural phosphorylated TK substrates.23 Table 1. Performances of electrochemical TK activity detection in the presence of free, adsorbed or immobilized TK and TK substrates in 50mM Hepes pH 7.0 (and 100 mM KCl) at 25 °C, 1 mM MgCl2, 0.1 mM

Reaction

Optimization was performed using signal/noise ratios. To select the suitable quantity of TK for these enzymatic activity measurements, the current intensity was recorded by using 0–13.9 µg (0–95.9 pmol) of TK (Supporting information S3a). The background current (noise, N) was measured with the reaction buffer containing 1 mM MgCl2, 0.1 mM Fe(CN)63-, 0.01 mM of TPP and various quantities of TK. The reaction was initiated by the addition of 5 µL of 6 mM L-ery. After 5 min of reaction, the anodic current was recorded at +0.56 V vs. Ag/ AgCl (signal, S). The highest S/N ratio (S/N = 6) is obtained with 13.9 µg of TK and this quantity was selected for further experiments. It is important to underline that this assay uses a low amount of TK in comparison to the spectrophotometric method described by Kochetov which uses around 200 mg of enzyme for a single assay28. The influence of Fe(CN)63-concentration was also studied (Supporting information S3b). S but also N increases with Fe(CN)63- concentration leading to the highest S/N using 0.1 mM of Fe(CN)63-. In parallel, oxidation of the product (here, glycolaldehyde if L-ery is used as donor substrate) by Fe(CN)63- was verified, meaning that the electrochemical response is only due to the oxidation of DHETPP. In these conditions, electrochemical responses to different TPP (Figure 1) and L-ery concentrations (Supporting information S3c) were obtained and the maximal current density (Jmax), the apparent Michaelis Menten constant (KM app, corresponding to the substrate concentration for which the current density is equal to Jmax/2), the linear range and the sensitivity were determined (Ta-

LR -2

Sensitivity -1

-2

KM app

(µA.cm )

(µM)

(mA.M .cm )

(µM)

Free TK

8.4 ± 0.1

0.5-20

166 ± 12

15

Adsorbed TK

8.0 ± 0.6

1-10

129 ± 17

TK@LDH

8.3 ± 0.4

1-20

69 ± 18

37

[b]

8.4 ± 0.4

1-150

32 ± 1

156

[b]

3.7 ± 0.7

5-100

25 ± 4

43

2.8 ± 0.2

1-100

14 ± 2

100

TPP Figure 1. Response curve for the detection of TPP: Experiments were performed using 13.9 µg of TK, 1 mM MgCl2, 0.1 3mM Fe(CN)6 , 1 mM L-ery in 0.05 M Hepes pH 7.0, 0.1 M KCl at 25 °C (n=3).

[c]

Jmax

[a]

33

Free TK L-ery

HPA

D-F6P

[b]

Fe(CN)63-, E=+0.56 V vs. Ag/AgCl. [a] in the presence of 1 mM L-ery, [b] in the presence of 0.2 mM TPP. HPA: hydroxypyruvic acid, D-F6P: D-Fructose-6-phosphate, [c] LR: Linear range

Immobilization of TK at the electrode surface could improve the performances for the screening assay. Recently, it was shown that the immobilization of TK can be carried out using layered double hydroxides (LDH) as immobilization matrix.34,45–47 These inorganic lamellar materials have porosity, biocompatibility and ions exchange properties suitable for enzyme immobilization.48 Here, same amount of TK (13.9 µg) was absorbed alone (adsorbed TK) or co-adsorbed with LDH nanoparticles (TK@LDH 1:1) at the surface of the electrodes and sensitivities for TPP detection were determined. Sensitivities are lower with adsorbed TK or TK@LDH (129 mA.M-1.cm-2 and 69 mA.M-1.cm-2 respectively) compared to free TK (166 mA.M-1.cm-2). As the immobilization does not offer improvements in this assay, this inhibition screening will be performed with free TK. Finally, as all compounds from chemical library are stored in neat DMSO, the electrochemical response was measured in the presence of 0-20 % (v/v) DMSO (Supporting information S4). S/N ratio increased up to 10 % (v/v) of DMSO and decreased beyond. Screening will be per-



formed in 10% (v/v) DMSO to ensure inhibitor solubility without affecting significantly the assay. This electrochemical assay allows to detect TK activity in only 7 minutes (5 minutes of reaction and 2 minutes of measurement) and the electrochemical device allows to perform 96 measurements simultaneously. 3.

Reproducibility and proof of concept with known inhibitors

To validate our high throughput electrochemical assay, a statistical parameter, namely Z-factor40, was used. Control and assay measurements were performed on 40 electrodes for the calculation of the Z-factor. The direct detection of Fe(CN)64- was used to evaluate the variability between the electrodes (Figure 2, black and white circles). A Z-factor of 0.64 (> 0.5) shows that the quality of electrodes is sufficient to detect Fe(CN)64-. To determine if the electrochemical assay can be used for TK inhibitor screening, the Z-factor obtained with the enzymatic reaction using 0.01 and 0.2 mM TPP was also determined (Figure 2, black, white and red squares). Z-factor value is 0.5 using saturated concentration of TPP (0.2 mM) related to an excellent assay according to Zhang et al.40 In these conditions, inhibition screening will allow to identify non-competitive inhibitors of TPP. However, Z-factor is close to zero in the presence of lower concentration of TPP (0.01 mM close to the KM app determined previously), related to a yes/no type assay. Despite of this lower Zfactor, the simplicity and rapidity of this electrochemical assay will allow to identify competitive inhibitors of TPP.

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iamine, pyrophosphate which are analogues of TPP and also with hydroxyphenylpyruvate which is an analog of the donor substrate. Oxythiamine was found be the best inhibitor with 84% of inhibition. Inhibition was also observed with all compounds except with hydroxyphenylpyruvate (HPP). In this special case, the current measured in the presence of HPP is not representative of TK response because this molecule is oxidized at the electrode, leading to an important noise. Cyclic voltammetry of HPP was performed between 0 and +1 V vs. Ag/AgCl (Supporting information S5a) showing that it is oxidized at +0.57 V vs. Ag/AgCl, close to the Fe(CN)64- oxidation potential. Therefore, compounds from chemical library which are sensitive to electrochemical oxidation will be excluded from the start.

Figure 3. Electrochemical detection of TK activity in the presence of 1 mM of thiamine (Th) oxythiamine (OxyTh), pyrophosphate (PPi) or hydroxyphenylpyruvate (HPP) and 0.01 mM TPP, 1 mM MgCl2, 13.9 µg TK, 1 mM L-ery, 0.1 mM 3Fe(CN)6 (in 0.05 M Hepes buffer pH 7.0 0.1 M KCl). The blank was obtained in the absence of TPP and the positive control with 0.01 mM of TPP and in the absence of inhibitor (n=3). The star indicates a signal which is not representative to TK response due to inhibitor oxidation at the electrode.

4.

High throughput screening and hit characterization

4-

Figure 2. Z-factor for the direct detection of Fe(CN)6 and for TK electrochemical responses. Measurements on 40 different electrodes were performed in the presence of 0.05 M Hepes buffer pH 7.0 with 0.1 M KCl (black circles, control) ; 0.1 mM 4Fe(CN)6 (white circles, assay) ; reaction media without TPP (black squares, control) and with 0.01 mM TPP (red squares, assay) or 0.2 mM TPP (white squares, assay). Current densities were sorted in descending order.

Before screening the chemical compounds library, a proof a concept was achieved with known inhibitors of TK from S. Cerevisiae (Figure 3). Electrochemical response was measured in the presence of 1 mM of thiamine, oxyth-

This electrochemical assay allows to perform 96 TK activity measurements in only 7 minutes, meaning that more than 800 samples can be assayed per hour. It can be easily used for the screening of TK inhibitors, particularly competitive inhibitors to the cofactor TPP. Taking into account positive and negative controls, 88 molecules from a chemical library can be assayed simultaneously. A total of 1360 molecules of the chemical library of ICBMS were assayed at a final concentration of 100 µM to identify new TK inhibitors. Electrochemical response was measured in the presence of TK, 0.01 mM TPP (close to its apparent Michaelis constant), 1 mM L-ery (saturating donor concentration) and each compound was assayed 4 times.


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The TPP concentration was chosen closed to the apparent Michaelis constant, a substrate concentration at which all kind of inhibitors (competitive, non-competitive or uncompetitive) shows significant effect (Supporting information S6).49 The percentage of TK inhibition was compared to controls obtained without inhibitor. A molecule was considered as a hit if it leads to a percentage of inhibition higher than 50%. On a total of 1360 molecules, 65 were excluded because of their electrosensitivity (confirmed by a cyclic voltammetry) and 110 molecules exhibited more than 50% inhibition in the first set. A second set of experiments was carried out with these hits but only one, namely I38-49, was confirmed to be an inhibitor with more than 80% inhibition at a concentration of 100 µM. This inhibitor, the 2-(4-ethoxyphenyl)-1-(pyrimidin-2yl)-1H-pyrrolo[2,3-b]pyridine (Table 2, I38-49) was synthetized according to Dimanche-Boitrel et al. to characterize the inhibition mechanism more deeply.41 An apparent IC50 of 63 µM was determined with the electrochemical assay (Supporting information S7) using inhibitor concentrations ranging from 0 to 300 µM and TPP concentration of 0.01 mM (apparent Michaelis constant). This value is lower than known TK inhibitors (280 µM for pyrophosphate18, 1400 µM for oxythiamin17, 34000 µM for thiamine18 and 3000 µM for hydroxyphenylpyruvate19). Moreover, cyclic voltammetry of this compound was performed to verify its electrochemical sensitivity (Supporting information S5b). Oxidation only occurred at potential higher than +0.8 V vs. Ag/AgCl (higher than the working potential of +0.56 V). Table 2. Influence of the chemical moieties on the percentage of inhibition of TK activity. The nature of the substituent R on the 2-(4-ethoxyphenyl)-1Hpyrrolo[2,3-b]pyridine scaffold is described on the following table.

Name of molecule

R

[a]

Percentage of [b] TK inhibition

I38-49

85%

(1)

23%

(2)

0%

(3)

0%

[a] The attachment point to the scaffold is indicated by an asterisk, [b] inhibition determined using a concentration of 100 µM.

An early lead compound has been identified by this screening and other substituted 2-(4-ethoxyphenyl)-1Hpyrrolo[2,3-b]pyridine closely related to I38-49, synthetized by Dimanche-Boitrel et al., are also present into the chemical library41. It offers the opportunity to identify key parameters of this scaffold that could be used for further inhibitors synthesis (Table 2). For example, if the pyrazine is replaced by a thiadiazole (compound (1)) only 23% of inhibition is obtained, and no inhibition if it is replaced by an electron-deficient heterocycle such as 5nitropyridine or a bulkier moiety like 4-(4pyrimidinyl)morpholine (compound (2) and (3)). These results suggests that, from a first set of compounds obtained from the library, the N1-substitution of the azaindole moiety 5 to 6-membered ring heterocycle is required to inhibit TK. Finally, kinetic parameters have been studied in the presence of different concentrations of I38-49. Classical methods for TK activity measurements were considered. The use of the spectrophotometric assay with alcohol dehydrogenase24 or galactose oxidase33 was impossible due to the inhibition of the secondary enzyme by I38-49 showing that this first lead compound lacks specificity. Moreover, the colorimetric assay based on tetrazolium red26 leads to high background with enzymatic substrates and products. To rapidly get first ideas about the inhibition mechanism and access to apparent kinetic parameters, IPA was used to measure TK activity in the presence of I38-49. Different inhibition mechanisms (Supporting information S8) were fitted to these curves. The inhibition mechanisms were simplified to the first step of the reaction, i.e. the fixation of TPP on TK (under L-ery saturation). The discrimination mode of Dynafit software was used to select the most appropriate inhibition mechanism thanks to the Akaike information criterion (AIC).42 With this method, it appears that I38-49 inhibits TK according to a partial non-competitive mechanism (Scheme 3): TK or TK-TPP forms can both bind I38-49 with similar KI (3.4 ± 0.9 µM) suggesting that the binding of TPP does not affect the inhibitor binding. On the other way, the binding constant of TPP onto the TK-I complex (KS = 22 ± 3 µM) is 4 times higher than the binding of TPP onto TK alone (KS = 4.8 ± 0.7 µM), suggesting that the inhibitor binding affects the binding of TPP. Nevertheless, both TK-TPP and TK-TPP-I are productive complexes with similar kinetic constants, including catalytic constant of enzymatic reaction and chemical constant for DHETPP oxidation (0.022 ± 0.001 min-1 and 0.018 ± 0.001 min-1). TK is not known to be an allosteric enzyme but Meshalkina et al.50 report that both TPP binding sites of TK have different affinities. A KI of 3.4 µM was found with this model which is remarkably low in comparison to known inhibitors. It is worth to note that no significant timedependent inhibition was observed (in a time scale of 2 hours) confirming the reversibility of the inhibition mechanism (Supporting information S9).



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AUTHOR INFORMATION Corresponding Author Scheme 3 : Proposition of the non-competitive inhibition mechanism of TK by I38-49. KS and KS2 are binding constants and kR and kR2 are overall kinetic constants (including catalytic constant of enzymatic reaction and chemical constant for DHETPP oxidation). More complex analyses such as the determination of the enzyme-inhibitor structure, molecular docking or Xray analysis are required to fully understand this mechanism together with accurate kinetic analysis. This inhibitor is an early lead compound that could be further improved to increase its specificity to reach the KI values in the nM range.

Conclusions Earlier works are related to the research of TK inhibitors for different therapeutic reasons but only few papers describe some efficient inhibitors with low IC50 or KI. A new high-throughput electrochemical assay was proposed here to quickly detect TK activity by oxidizing the reaction intermediate. The chemical library of ICBMS was screened (1360 molecules) allowing to identify an early lead compound for inhibitors of E. Coli TK. The electrochemical assay also allows to suggest a partial noncompetitive mechanism related the complex TK mechanism. A structural optimization has to be performed to increase the specificity and the efficiency of this inhibitor but the importance of the N1-substitution of azaindole ring in this first scaffold could be emphasized. This assay will be transposed to other therapeutically relevant TKs to identify disease specific inhibitors. On the contrary to other enzymatic methods, this rapid and sensitive assay requires only one donor substrate, a low amount of enzyme and is not sensitive to optical interferences. Moreover, no auxiliary enzyme is required, these ones requiring a pH compatible with TK activity and potentially inhibited by the screened compounds. This pH-independent electrochemical assay could be adapted to any TPP-dependent enzymes.

ASSOCIATED CONTENT

Bastien Doumèche. Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, ICBMS UMR 5246 CNRS, Université de Lyon, Université Lyon 1, CNRS, INSA Lyon, CPE Lyon. 43 bd du 11 Novembre 1918, 69622 Villeurbanne Cedex (France). E-mail: [email protected]. Franck Charmantray Institut de Chimie de Clermont Ferrand, ICCF UMR 6296 CNRS-UCA-Sigma F-63000 ClermontFerrand (France). E-mail: [email protected]

Author Contributions All authors have given approval to the final version of the manuscript.

Funding Sources The French Ministry of Science and Education is gratefully acknowledged for funding and the authors would like to thank the French ANR agency (ANR 13-JSV5-0002-01 Transbioscreen).

ABBREVIATIONS AIC, Akaike information criterion; DHETPP, α,βdihydroxyethylthiamine diphosphate; HPP, hydroxyphenylpyruvate; IPA, intermittent pulse amperometry; LDH, layered double hydroxides; L-ery, L-erythrulose; PPP, pentose phosphate pathway; TK, transketolase;

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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures: production, purification and activity test for TK from E. Coli, synthesis of I38-49, characterization of the electrode, comparison of analytical performances of TK-based assays, calibration curve of 4Fe(CN)6 , parameters optimization, influence of DMSO on the activity measurements, cyclic voltammetry of hydroxyphenylpyruvate and of I38-49, theoretical IC50 for classical inhibitors, apparent IC50 of I38-49, enzyme mechanisms discrimination for the Dynafit Software and time-dependent TK deactivation.

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Innovative electrochemical screening allows to identify new

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