Mamadou Daffé
email: mamadou.daffe@ipbs.fr

Hedia Marrakchi
email: hedia.marrakchi@ipbs.fr

Ségolène Galandrin

Valérie Guillet

Rajendra S Rane

Mathieu Léger

Nathalie Eynard

Kaveri Das

Tanjore S Balganesh

Lionel Mourey

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

Keywords: mycobacteria, fatty acyl-AMP ligase, FAAL, assay adaptation, screening of inhibitors reduction

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of T m (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

Introduction

Tuberculosis (TB) remains the leading cause of mortality due to a single infectious agent and, in conjunction with the spread of human immunodeficiency virus (HIV) infection, is today among the worldwide health threats. Emergence of "MDR" (multidrug-resistant) and "XDR" (extensively drug-resistant) Mycobacterium tuberculosis (Mtub) strains is partly responsible for the treatment failure and for the recrudescence of this disease. The development of new antibiotics effective against resistant strains is one of the major priorities of worldwide programs of the World Health Organization 1 to fight against TB. [START_REF] Ma | Global Tuberculosis Drug Development Pipeline: The Need and the Reality[END_REF] Mycobacteria produce a remarkable collection of lipophilic molecules ranging from simple short-chain fatty acids to the very complex long-chain mycolic acids. [START_REF] Marrakchi | Comprehensive Overview of Mycolic Acid Structure and Biosynthesis[END_REF][START_REF] Gokhale | Versatile Polyketide Enzymatic Machinery for the Biosynthesis of Complex Mycobacterial Lipids[END_REF] Among the unique lipids of Mtub, being the main constituent of the outer membrane, mycolic acids play a critical role in both the architecture and permeability of the mycobacterial cell envelope. [START_REF] Zuber | Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State[END_REF][START_REF] Sani | Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins[END_REF] The biosynthesis of these exceptionally long (C 70 -C 90 ), alpha-branched, beta-hydroxylated fatty acids is essential for the survival of Mtub and provides thereby opportunities to target numerous enzymes of this metabolic pathway. The synthesis of mycobacterial lipids 474691J BXXXX10.1177/1087057112474691Jour nal of Biomolecular ScreeningGalandrin et al. 2013 requires the combined action of fatty acid synthases (FASs) and polyketide synthases (PKSs). The mycolic acid synthesis involves both FAS-I (for de novo synthesis) and FAS-II (for elongation), as well as the action of Pks13 to achieve the final condensation between two activated fatty acids (Fig. 1A). A specific subclass of the FadD (fatty acid activating) family of enzymes establishes the crosstalk between FASs and PKSs by providing the activated fatty acyl adenylates to their cognate PKSs. [START_REF] Gokhale | Versatile Polyketide Enzymatic Machinery for the Biosynthesis of Complex Mycobacterial Lipids[END_REF] The Mtub genome contains 34 fadD genes encoding for these FadD proteins grouped in two subclasses: fatty acyl-CoA ligases (FACLs) involved in lipid degradation and fatty acyl-AMP ligases (FAALs) dedicated to lipid biosynthesis. [START_REF] Trivedi | Enzymic Activation and Transfer of Fatty Acids as Acyl-Adenylates in Mycobacteria[END_REF] The FAALs activate and transfer fatty acids to PKSs for further chain extension to provide most of the highly specialized lipids of Mtub. Many of the Mtub FadD proteins represent potential drug targets due to their confirmed essentiality or requirement for virulence. [START_REF] Duckworth | Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets[END_REF] Several FAAL functions have been characterized and shown to activate carboxylic acid substrates and load them onto cognate PKS proteins. For instance, FAAL22, which loads onto Pks15/1, was demonstrated to be essential for the synthesis of the virulence-conferring phenolic glycolipids (PGLs), [START_REF] Ferreras | Mycobacterial Phenolic Glycolipid Virulence Factor Biosynthesis: Mechanism and Small-Molecule Inhibition of Polyketide Chain Initiation[END_REF] whose synthesis also involves FAAL29, which loads onto the polyketide synthases PpsA-E. [START_REF] Simeone | Delineation of the Roles of FadD22, FadD26 and FadD29 in the Biosynthesis of Phthiocerol Dimycocerosates and Related Compounds in Mycobacterium tuberculosis[END_REF] Similarly, FAAL26, which loads onto PpsA-E, and FAAL28, which loads onto Mas, are involved in the biosynthesis of the phthiocerol and mycocerosate, respectively, of the phthiocerol dimycocerosates (DIMs) virulence factor. Likewise, FAAL23, which loads onto Pks2, is implicated in the synthesis of sulfolipids, whereas FAAL33 (also called MbtM), which loads onto MbtL, is involved in mycobactin synthesis.

One of the most extensively studied FAALs, probably because of its confirmed essentiality in mycobacteria, is FAAL32, more known as and thereafter named FadD32, required for mycolic acid synthesis. [START_REF] Portevin | The Acyl-AMP Ligase FadD32 and AccD4-Containing Acyl-CoA Carboxylase Are Required for the Synthesis of Mycolic Acids and Essential for Mycobacterial Growth: Identification of the Carboxylation Product and Determination of the Acyl-CoA Carboxylase Components[END_REF] FadD32 acts in concert with Pks13 and activates the very long meromycolic acid (C 50 -C 60 ) prior to its condensation with a C 24 -C 26 fatty acid, [START_REF] Gavalda | The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis[END_REF] which itself is activated by the AccD4-containing acyl-CoA carboxylase ACCase, to yield, upon reduction, mycolic acids (Fig. 1A). [START_REF] Portevin | The Acyl-AMP Ligase FadD32 and AccD4-Containing Acyl-CoA Carboxylase Are Required for the Synthesis of Mycolic Acids and Essential for Mycobacterial Growth: Identification of the Carboxylation Product and Determination of the Acyl-CoA Carboxylase Components[END_REF][START_REF] Portevin | A Polyketide Synthase Catalyzes the Last Condensation Step of Mycolic Acid Biosynthesis in Mycobacteria and Related Organisms[END_REF][START_REF] Gande | Acyl-CoA Carboxylases (accD2 and accD3), Together with a Unique Polyketide Synthase (Cg-pks), Are Key to Mycolic Acid Biosynthesis in Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis[END_REF] The operon fadD32-pks13-accD4, present in all the mycobacterial species and Mtub strains sequenced so far, was proved to be essential for the viability of mycobacteria. [START_REF] Portevin | The Acyl-AMP Ligase FadD32 and AccD4-Containing Acyl-CoA Carboxylase Are Required for the Synthesis of Mycolic Acids and Essential for Mycobacterial Growth: Identification of the Carboxylation Product and Determination of the Acyl-CoA Carboxylase Components[END_REF][START_REF] Portevin | A Polyketide Synthase Catalyzes the Last Condensation Step of Mycolic Acid Biosynthesis in Mycobacteria and Related Organisms[END_REF] Conditional expression of fadD32 has confirmed its essentiality for the growth of Mtub, [START_REF] Boldrin | Development of a Repressible Mycobacterial Promoter System Based on Two Transcriptional Repressors[END_REF][START_REF] Forti | Pristinamycin-Inducible Gene Regulation in Mycobacteria[END_REF] Mycobacterium smegmatis (Msmeg), [START_REF] Portevin | The Acyl-AMP Ligase FadD32 and AccD4-Containing Acyl-CoA Carboxylase Are Required for the Synthesis of Mycolic Acids and Essential for Mycobacterial Growth: Identification of the Carboxylation Product and Determination of the Acyl-CoA Carboxylase Components[END_REF] and Mycobacterium abscessus, [START_REF] Cortes | Conditional Gene Expression in Mycobacterium abscessus[END_REF] whereas knockdown studies have established that the fadD32 operon is a vulnerable target. [START_REF] Carroll | Identifying Vulnerable Pathways in Mycobacterium tuberculosis by Using a Knockdown Approach[END_REF] Accordingly, FadD32 represents a particularly attractive drug target.

Substrate analogues as inhibitors of FadD32 have been characterized using a radiometric assay coupled with thinlayer chromatography (TLC). [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] However, limited throughput of this assay is not suitable for screening large libraries of compounds to find novel inhibitors. We describe, in the present study, the development and the optimization of a nonradiometric assay for FadD32 activity using the Msmeg FadD32 orthologue as a surrogate enzyme source. The adapted assay was used to screen a selected library, which led to the identification of five novel classes of inhibitors.

Materials and Methods

Cloning of the fadD32 Orthologous Genes

The fadD32 genes were PCR-amplified from total DNA of Msmeg mc²155 and Corynebacterium glutamicum (Cglu) 13032 strain using the high-fidelity Pfu DNA polymerase (Fermentas, Vilnius, Lithuania) with the following specific primers (Sigma-Aldrich, St. Louis, MO):

Msmeg fadD32 5′ (NdeI) GCACATATGATGCCGTT CCACAATCCG Msmeg fadD32 3′ (NdeI) GCACATATGATGGCGTT GTTGGTCAGTC Cglu fadD32 5′ (NdeI) GCGTCGGCGCATATGAGAG GGGTTCACATGGA Cglu fadD32 3′ (BamHI) ACGGATCCCTGCGGTGTCT GCAAAGA
The PCR fragments were ligated into the pET15b expression vector (Novagen/Merck, Darmstadt, Germany) digested with appropriate restriction enzymes (Fermentas) and dephosphorylated to avoid recircularization (CIAP; Fermentas). Calcium-competent Escherichia coli cells (DH5α; Invitrogen, Paisley, UK) were transformed with ligation products, and carbenicillin-resistant (50 µg/mL carbenicillin; Sigma-Aldrich) clones were selected. DNA sequencing (MilleGen, Toulouse, France) of the cloned fragments after plasmid preparation (Qiagen, Valencia, CA) confirmed the correctness of the sequences.

Protein Expression and Purification

Competent E. coli BL21 Star (DE3) One Shot (Invitrogen) were transformed with the pET15b-fadD32 plasmid, [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] and expression of FadD32 proteins was performed using autoinducible medium as described previously by Studier. [START_REF] Studier | Protein Production by Auto-induction in High Density Shaking Cultures[END_REF] Briefly, a starter culture grown overnight in LB medium was diluted in auto-induction medium. [START_REF] Studier | Protein Production by Auto-induction in High Density Shaking Cultures[END_REF] Cells were grown for 72 h at 20 °C and collected by centrifugation at 4 °C for 15 min. The pellet was then resuspended in lysis buffer consisting of 50 mM HEPES (pH 7.5), 10% glycerol (v/v), 30 mM imidazole, 500 mM NaCl, 0.75 mg/mL lysozyme, and 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; Sigma-Aldrich) and frozen overnight at -80 °C. The frozen bacterial pellets were thawed at room temperature (RT) and disrupted by sonication (four intermittent pulses of 30 s) on a VibraCell (Fisher Bioblock Scientific, Illkirch, France) and centrifuged at 20 000 g for 30 min at 4 °C. The clarified lysate was loaded onto an HisTrap HP (1-mL) affinity column (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and FadD32 protein was eluted using 150 mM imidazole in 50 mM HEPES (pH 7.5), 500 mM NaCl, and 0.2 mM AEBSF. The fractions containing FadD32 protein were pooled and concentrated using Vivaspin 2 (10 000 Da molecular weight [MW] cutoff; Sartorius, Goettingen, Germany) and loaded onto a gel filtration column (HiLoad 16/60 Superdex 200; GE Healthcare) preequilibrated with 50 mM HEPES (pH 7.5), 500 mM NaCl, and 0.2 mM AEBSF. The purified proteins (as checked on sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE] followed by a Coomassie blue staining) were then concentrated and stored at -20 °C in 50% glycerol.

R 1 CO AMP R 1 CH COOX R 2 CH OH R 2 CO SCoA CH COOH Carboxyacyl-CoA

FA synthesis

Long-chain FA activation

Mycolic acid condensation

Radiometric-TLC Enzyme Assays

The formation of the acyl-AMP product by FadD32 from fatty acid and adenosine triphosphate (ATP) in the presence of Mg 2+ was monitored by radiolabeled-TLC (radio-TLC), as described previously. [START_REF] Trivedi | Enzymic Activation and Transfer of Fatty Acids as Acyl-Adenylates in Mycobacteria[END_REF][START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] Briefly, the reaction mixtures were performed as described for spectrophotometric assays, in 30 µL in glass microtubes, and incubated for 80 min at RT. Reactions were then stopped by adding 5% final acetic acid. The reaction mixture was then spotted in duplicates on silica gel TLC plates (G60; Macherey-Nagel, Düren, Germany), with the radiolabeled fatty acid substrate being subsequently separated from the acyl-AMP product on the basis of their differential R f (frontal ratio). Radiolabeled products were resolved in butan-1-ol/acetic acid/water (80:25:40, v/v/v) at RT and visualized by exposing the TLC to a Fujifilm (Tokyo, Japan) imaging plate prior to phosphorimager detection (Typhoon Trio; GE Healthcare). The percentage of product was calculated after quantitation of both fatty acid and acyl-AMP using the ImageQuant software version 5.1 (Molecular Dynamics, Sunnyvale, CA).

Spectrophotometric Enzyme Assays

FadD32 enzyme catalyzes the formation of acyl-AMP using fatty acid and ATP as substrates (Fig. 1B). Pyrophosphate (PPi) released during this reaction was hydrolyzed using a Pyrophosphatase coupled reaction, and the resulting inorganic phosphate (Pi) was measured using a colorimetric assay. Addition of Malachite Green reagent to the reaction mix resulted in the formation of phosphomolybdate complex with an increase in absorbance at 630 nm (A 630 ) (Fig. 1B). All assays were performed in quadruplicates in 384-well polystyrene microplates (Greiner Bio One, Courtaboeuf, France), using the PiColorLock Gold kit (Innova Biosciences, Cambridge, UK). Reactions were conducted at RT, in 30 µL of assay mix containing 50 mM HEPES (pH 7.5), 8 mM MgCl 2 , 0.01% Brij-58, 2 mM ATP, 20 µM lauric acid, 1 mM DTT (1,4-dithiothreitol), and 2 mU/mL Pyrophosphatase (Sigma-Aldrich). Reactions were initiated by the addition of 15 µL FadD32 enzyme diluted in 50 mM HEPES (pH 7.5) to 15 µL of 2× assay mix. After 80 min, reactions were stopped by adding 30 µL of cold reaction buffer and 15 µL of Malachite Green. The A 630 was read after 5 min of incubation at RT in a µQuant counter (Bio-Tek, Winooski, VT). A reaction without enzyme (for protein dose curve experiments) or without substrate (for K m and V max determinations) was performed in each experiment and used as blank. To calculate the concentration of Pi, a calibration curve using known concentrations of Pi varying from 10 to 80 µM was performed in each experiment according to the manufacturer's recommendations.

For apparent kinetic parameters (K m , V max , and k cat ) determination, the initial velocity was measured (incubation time of 40 min) as a function of the studied substrate concentration at saturating concentrations of the nonvaried substrate. The kinetic parameters were determined when initial velocities were measured at a 90-min incubation time and found identical to those calculated at 40 min. The saturation curve was fit by nonlinear regression analysis using the following GraphPad Prism 4.02 (GraphPad Software, La Jolla, CA) equation:

Vi = V max * [S]/(K m + [S])
, where V max is the maximal velocity, [S] is the substrate concentration, and K m is the Michaelis-Menten constant. Kinetic parameters for fatty acids were determined at fixed concentrations of ATP (2 mM) and by varying concentrations of the fatty acid (3.1-200 µM), whereas kinetic constants for ATP were determined at fixed concentrations of lauric acid (C 12 ) (200 µM) and by varying concentrations of ATP (0.05-4 mM) (Suppl. Fig. S1). The catalytic constant (k cat ) reflects the maximum rate of product formation and was calculated by dividing V max by the total enzyme concentration

k cat = V max /[E T ].
The [E T ] was 1.2 pmol in the 30-µL assay reaction, and V max was inferred from the Michaelis-Menten equation (described above and in Suppl. Fig. S1).

For inhibition studies, concentrations of ATP and lauric acid were adjusted to be above or around the K m (0.25 mM ATP and 20 µM lauric acid). The substrate analogue alkyladenylate AMPC12 (chemically synthesized as described previously [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] ) was first diluted in DMSO, and the substrate mixture was applied to increasing concentrations of the compound. The reaction was started by adding the enzyme. Data were expressed as percentage of inhibition. Curve fitting and EC 50 calculations were performed using sigmoidal dose response as the equation model (GraphPad Prism 4.02 software).

Thermostability of FadD32

The melting point (T m ) of FadD32 was measured by differential scanning fluorimetry (DSF) and was used both to characterize the enzyme thermal stability under various conditions (buffer, pH) and to identify ligands or inhibitors. [START_REF] Pantoliano | High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery[END_REF] A mix of enzyme (4 µM), SYPRO orange (5×) (Invitrogen), buffer (100 mM), and NaCl (500 mM) was exposed to a temperature gradient from 25 to 80 °C with a 0.3 °C increment. All measurements were performed in triplicates in 96-well plates (Biorad, Marnes-la-Coquette, France). The thermal transition was monitored using an RTQPCR CFX96 Real-Time System (Biorad). T m was given by the inflexion point of the curve RFU (relative fluorescence unit) = f(T). Before fluorimetry experiments, the FadD32 samples were soaked in the presence of substrate analogue (AMPC12) or in buffer at a final concentration of 40 µM. For the DSF experiments, the His-tag of the affinity-purified FadD32 was removed by thrombin cleavage (Novagen). The protein solution (after 5-fold dilution to reduce the imidazole concentration down to 30 mM) was submitted to a 3-h cleavage reaction at RT with 0.28 U/mL of thrombin. The cleaved protein was then reloaded onto a HisTrap HP (1-mL) affinity column to eliminate the noncleaved fraction. The flow-through protein-containing fractions were then pooled, concentrated, and purified by size exclusion chromatography onto a Superdex 200 Prep Grade column (GE Healthcare). Samples used for DSF experiments were stored at -80 °C without glycerol.

Biochemical High-Throughput Screening

The above-mentioned biochemical assay to measure FadD32 activity was adapted for high-throughput screening (HTS). All the chemicals and biochemicals were procured from Sigma-Aldrich unless otherwise stated. The MsmegFadD32 enzyme was prepared as described above. The 30-µL reactions containing 50 mM HEPES buffer (pH 7.7), 0.25 mM ATP, 8 mM MgCl 2 , 0.001% Brij-35P, 1 mM DTT, 0.1 U/mL Pyrophosphatase, 20 nM MsmegFadD32 enzyme, and 20 µM lauric acid were carried out in 384-well microplates (Corning Costar; Corning Life Sciences, Tewksbury, MA) at room temperature (25 °C) for 100 min. Then, 30 µL of modified Baykov's Malachite Green reagent [START_REF] Baykov | A Malachite Green Procedure for Orthophosphate Determination and Its Use in Alkaline Phosphatase-Based Enzyme Immunoassay[END_REF] was added and mixed for color development. Assay additions and mixing were done using the Apricot PP-384-M Liquid Handling System (Apricot Designs, Covina, CA). The plates were further incubated at 25 °C for 20 min. At the end of this incubation time, the plates were read at a 635-nm wavelength in a Spectramax 384 Plus microplate reader (Molecular Devices, Sunnyvale, CA). The single-point primary screening and the 10-point concentration response (CR) studies were performed using this assay. The inhibition was calculated against the following reaction controls: MIN (0% reaction control) = 1 µL of 0.5 M EDTA (16.7 mM final) and MAX (100% reaction control) = 1 µL of 100% DMSO (3.3% final). Since the enzymatic mechanism process for the two substrates is yet unknown, the concentrations of ATP and lauric acid were adjusted to be above or around their respective K m to select moderate to weak inhibitors of both substrates.

In the primary screening, 10 177 compounds were screened in singlicates at 20 µM. These compounds were randomly selected from a large, chemically diverse library of the AstraZeneca Corporate Compound collection to check the feasibility of HTS. This collection was made by acquiring chemically diverse sets of compounds from various sources as well as by adding compounds synthesized during various drug projects within AstraZeneca.

Promiscuity Test

High protein effect (promiscuity) assays were done to ascertain high protein (MsmegFadD32) effect on the compound CR. Basically, the above-mentioned high-throughput biochemical assay was done for a given set of compounds simultaneously at 1× (20 nM) and 5× (100 nM) enzyme concentrations and with a 10-point CR similar to the assay described before, except that the reaction time was reduced to 20 min to maintain the reaction linearity with increased enzyme concentration. [START_REF] Feng | A Detergent-Based Assay for the Detection of Promiscuous Inhibitors[END_REF]

Thermofluor (T m Shift) Assay

The Thermofluor assay was performed as described before [START_REF] Venkatraman | Screening, Identification, and Characterization of Mechanistically Diverse Inhibitors of the Mycobacterium tuberculosis Enzyme, Pantothenate Kinase (CoaA)[END_REF] to ascertain binding of these compounds to MsmegFadD32 enzyme. Thermal stability measurements were recorded in 96-well PCR plates covered with optical film (Bio-Rad, Hercules, CA), using a real-time PCR thermal cycler (iQ5 from Bio-Rad). The environmentally sensitive dye, SYPRO Orange (Invitrogen), was used to monitor protein unfolding. Thermal unfolding was monitored at 0.5 °C intervals from 25 °C to 95 °C. The assay was performed at the optimal reaction buffer in a 50-µL volume (50 mM HEPES [pH 7.7], 8 mM MgCl 2 , 6.25× SYPRO Orange, and 0.15 mg/mL MsmegFadD32). The final DMSO concentration was maintained at 4% (v/v). Under these conditions, ATP, nonhydrolysable ATP analogue adenosine 5′-[γ-thio] triphosphate (ATP-γ-S) (Sigma-Aldrich) and lauric acid were tested for binding to MsmegFadD32 at 1× and 5× concentrations of their respective K m values. All 32 compounds selected after hit evaluation were tested in this assay for binding to MsmegFadD32 at three concentrations (12.5, 25, and 50 µM) (Suppl. Table S1A). The 16 "nonpromiscuous" compounds were assessed for the competition with ATP-γ-S (Suppl. Table S1B).

Results and Discussion

Enhancement of the FadD32 Purification Yield

Previously, we successfully expressed and purified the MtubFadD32 (MtubFadD32), yet with a relatively low protein yield (0.7 mg/L of culture). [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] To improve the MtubFadD32 expression and/or solubility, we reasoned that expression host strains reported to allow stabilization of mRNA levels of recombinant proteins should allow improved protein expression levels and stability, whereas auto-induction media should slow down the expression rate while decreasing handling of cultures. Accordingly, the MtubFadD32 protein was expressed by culturing Star One Shot (Invitrogen) BL21(DE3)/pET15b-MtubfadD32 in autoinduction media for 72 h at 20 °C. Using these culture conditions, the amount of MtubFadD32 protein recovered after purification using the reported protocols was more than 7 mg/L of culture, which represented at least a 10-fold increase in the protein yield compared with conditions where protein expression was performed in LB medium (5-h induction with 0.5 mM isopropyl-β-d-thiogalactopyranoside [IPTG] at 25 °C). [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] The protein obtained after the two-step purification was homogeneous (Suppl. Fig. S2).

FadD32 Assay Development

The MtubFadD32 activity was previously characterized using a radiometric assay coupled to thin-layer chromatography (radio-TLC). The search for FadD32 inhibitors through screening of chemical libraries requires the design of suitable readouts compatible with low volumes and robotics. The FadD32 protein displays FAAL activity in vitro, catalyzing the formation of an acyl-adenylate (acyl-AMP) from a fatty acid and ATP (in the presence of Mg 2+ ), with the release of PPi (Fig. 1B). We decided to couple the FadD32 reaction to inorganic Pyrophosphatase (iPPase), which cleaves the PPi into Pi, the latter being measurable with inorganic phosphate-interacting reagents such as Malachite Green, followed by spectrophotometric detection at 630 nm, as demonstrated for Pi-generating enzymes. [START_REF] Baykov | A Malachite Green Procedure for Orthophosphate Determination and Its Use in Alkaline Phosphatase-Based Enzyme Immunoassay[END_REF] The initial assays of FadD32 activity using the Pyrophosphatase-coupled reaction were performed in 384well plates in conditions similar to those applied in the radio-TLC assay previously reported, [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] except for the [1-14 C]C 12 fatty acid substrate, which was replaced by cold lauric acid (C 12 ) and the addition of Pyrophosphatase in the reaction mixture. The FadD32 reaction was followed at 630 nm, and a Pi standard curve allowed quantitation of the Pi released, thus enabling the pyrophosphate or acyl-AMP concentrations formed during the reaction to be deduced. To test the linearity of the product formation in these conditions, a protein dose-response curve (with increasing concentrations) of MtubFadD32 in the reaction was performed. For protein concentrations up to 600 nM in the assay, the increase in A 630 remained low, and at least 1.2 µM was needed to detect FAAL activity (Fig. 1D, dotted line).

Among the various parameters to be optimized to adapt the FadD32 assay to HTS are (1) the protein concentration, optimally in the nanomolar range, and (2) the substrate's (fatty acid) chain length and concentration, which should be kept to a minimum given its hydrophobic nature and low solubility.

The relatively high concentrations of MtubFadD32 added in the reaction seemed not compatible with the adaptation of the assay to HTS. We therefore considered using FadD32 orthologues as surrogates to develop an HTS assay, knowing that the use of the M. smegmatis orthologue in the mycobacterial PimB protein biochemical and structural studies has been shown to be valuable. [START_REF] Guerin | New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria: PimB′ is an essential enzyme of Mycobacterium smegmatis[END_REF] The FadD32 proteins being highly conserved in all mycolic acid-producing bacteria (grouped in the Corynebacteriales suborder), two FadD32 orthologues from Msmeg and Cglu-sharing, respectively, 75% and 40% sequence identity with the MtubFadD32-were selected as surrogate candidates for comparative biochemical and inhibition studies. The respective genes were cloned in pET15b expression vectors with an N-terminal 6xHistidine tag for production in E. coli strains. The three FadD32 proteins were expressed and purified (Fig. 1C) according to improved protocols of expression and purification mentioned above (see Materials and Methods). The protein expression rates and purification yields obtained for MsmegFadD32 and CgluFadD32 were significantly higher than those of MtubFadD32 (Table 1). Indeed, the calculated volumes of culture required for performing a significant number of assays clearly distinguished the MsmegFadD32 and CgluFadD32 from the Mtub protein and indicated that the former orthologues are by far more convenient for HTS purposes than the Mtub protein (Table 1).

When the thermal stability of the three FadD32 orthologues was investigated through determination of the melting temperature (T m ), MtubFadD32 was the least stable protein, with a T m of 37 °C, whereas MsmegFadD32 and CgluFadD32 were much more thermally stable in our experimental conditions, with a T m of 46 °C and 59 °C, respectively. The activities of the MtubFadD32, MsmegFadD32, and CgluFadD32 proteins were then measured in parallel in a protein dose-response experiment, and their abilities to release Pi in the FadD32-Pyrophosphatase coupled assay were compared (Fig. 1D). A linear relationship between velocity and enzyme concentration was observed throughout the enzyme concentrations tested. The specific activity of CgluFadD32 was only twice that of MtubFadD32 (Table 1). In sharp contrast, the dose-response curve for the MsmegFadD32 enzyme showed a linear response down to a few nanomolar of protein in the assay (Fig. 1D,inset). Indeed, the specific activity of MsmegFadD32 was approximately 75 times higher than that of the MtubFadD32 enzyme (Table 1). The CgluFadD32 displayed high thermal stability and purification yield, yet its low specific activity is not suitable for performing inhibition and HTS assays. Furthermore, when combining the purification yield, the amount of protein required in the assay, and enzyme activity for the three FadD32 orthologues (Table 1), the Msmeg protein displayed the lowest volume of culture necessary to perform a 100-microplate (384-well plates) assay, thus permitting the achievement of an HTS campaign with at BIBLIOTHEQUE INTERUNIV SANTE on January 30, 2013 jbx.sagepub.com Downloaded from a unique batch of protein. The higher sequence identity of MsmegFadD32 with the Mtub enzyme, combined with its favorable biophysical properties and high specific activity, made the MsmegFadD32 enzyme the most promising surrogate for conducting the assay development and optimization toward the HTS of inhibitors.

Assay Optimization Using FadD32 from M. smegmatis

To gain insight into the optimal assay conditions (i.e., reaction volume and composition), several parameters were investigated using MsmegFadD32. A reaction volume of 30 µL was used to reduce the amount of protein in the assay while keeping the most favorable optical performance. To keep the benefit of using larger volumes at the reading step, which allowed reducing reading errors due to meniscus curvature and shorter path length, [START_REF] Harrison | Location Dependent Biases in Automatic 96-Well Microplate Readers[END_REF] the reaction mixture was diluted 1:1 (v/v) postincubation with basal buffer before taking a readout. Indeed, higher ratios of signal/background (S/B of 4.2 vs. 2.6) and signal/noise (S/N of 97 vs. 23) compared with no buffer dilution were achieved (Suppl. Fig. S3A).

The critical parameters affecting the enzyme activity were the reaction buffer and pH. These were first investigated by inspecting the protein thermal stability using DSF, a widely used technique for screening optimal conditions for biological macromolecules. [START_REF] Pantoliano | High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery[END_REF] When thermal stability of the MsmegFadD32 protein was inspected at different buffer and pH conditions (from 5-8), a higher T m was observed at neutral pH, indicating an increased stability of the protein at this pH (Fig. 2A). Investigation of FAAL activity of MsmegFadD32 under the same conditions revealed that the use of acidic buffer (pH 5) in the reaction mixture did not allow detection of FAAL activity, even at a higher protein concentration in the assay (Fig. 2B). The highest protein activity was observed at neutral pH using HEPES buffer, with the pH 7.5 being the most suitable for further enzymatic assays (Fig. 2B, insert), in agreement with the thermal stability data.

A time course experiment of MsmegFadD32 activity was performed in the presence of three enzyme concentrations to determine the time window for reaction linearity. The reaction rate was linear between 40 and 120 min, ensuring initial velocity (V 0 ) conditions at all enzyme concentrations tested (Fig. 2C).

The low solubility of the fatty acids in the reaction mixture was a critical issue for the availability of the substrate in the FAAL reaction. Accordingly, the effects of detergents on the kinetic parameters of MsmegFadD32 (for lauric acid) were examined in the absence or presence of different agents. The presence of detergent in the assay significantly improved the reaction rate, with Brij (58 and 35) exhibiting the most pronounced effect (Suppl. Fig. S3B).

Since most of the library compounds are solubilized in DMSO, the effect of DMSO on MsmegFadD32 activity was investigated, and concentrations up to 7.5% did not appear to affect the activity of the enzyme (data not shown).

Enzymatic Characterization of M. smegmatis FadD32

The choice of the fatty acid substrate and determination of kinetic parameters for both substrates (ATP and fatty acid) of FadD32 are critical for the determination of the conditions to set for inhibition and screening experiments. The apparent K m , V max , and k cat were determined for ATP and for fatty acids of various chain lengths (Table 2). The myristic acid (C 14) displayed the highest catalytic efficacy (k cat /K m ), in agreement with the FadD32 substrate preference for long-chain fatty acids. [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] For further inhibition and HTS experiments, lauric acid (C 12) was chosen since it combines the advantage of having a relatively short aliphatic chain with a high catalytic efficacy. In addition, the K m value for C 12 permits including a concentration of the substrate in the assay close to the K m (20 µM), which would allow the detection of all types (competitive, uncompetitive, and noncompetitive) of inhibitors with a sufficient absorbance change. The lower K m of the C 14 could be a disadvantage if one is looking for competitive inhibitors, and the assay would require several-fold the K m of the substrate to generate sufficient signal. The K m of ATP measured in the assay conditions was 0.08 mM.

We evaluated the correlation between the release of Pi using the PiColorLock Gold (Innova Biosciences) Identity scores with the M. tuberculosis protein were computed from protein sequence alignment performed using the BLAST program (NCBI, Bethesda, MD). The protein purification yields are calculated per liter of culture expressing the protein of interest. The specific activity of each FadD32 was deduced from the linear regression protein dose-response curve (Figure 2B) (protein ranging from 1200 to 300 nM for MtubFadD32 and CgluFadD32 and from 100 to 3.125 nM for MsmegFadD32). The volume of culture required for performing a 100-microplate assay (384-well plates) was calculated based on addition of 1200 nM of MtubFadD32, 40 nM of MsmegFadD32, and 600 nM of CgluFadD32 protein per reaction.

detection and the formation of acyl-AMP determined by radio-TLC (Fig. 3). In the same conditions (except for the radiolabeled substrate), a protein dose-response curve experiment was performed, and the product formation was analyzed with either the radio-TLC assay (Fig. 3A) or the Pyrophosphatase-coupled FadD32 assay. The good correlation of the amount of product formed using both assays validated the PiColorLock as a reliable and quantitative assay for measuring FadD32 activity (Fig. 3B). Thus, the optimization of the assay conditions and its adaptation to HTS afforded a robust and time-saving assay, which also represents a powerful tool for studying the biochemical properties of this FAAL and others (determination of enzyme kinetic parameters, inhibition studies) and makes the assay an attractive option for the identification of FAAL inhibitors.

Binding of a Substrate Analogue and Inhibition of FadD32 Activity

The shift in the thermal stability of MsmegFadD32 in the presence of a substrate analogue (alkyl-adenylate, AMPC12), previously reported to inhibit the MtubFadD32 enzyme activity, [START_REF] Leger | The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis[END_REF] was investigated using T m measurements. The addition of AMPC12 to the protein induced a significant (6 °C) thermal shift (∆T m ) compared with the protein alone at pH 7.5 (Fig. 4A). This shift indicated that the inhibitor binds to the protein, and this interaction very likely stabilizes MsmegFadD32. The effect of AMPC12 on the protein activity was tested and, expectedly, a dose-dependent inhibition of the enzyme activity was observed in the presence of increasing concentrations of the alkyl-AMP (Fig. 4A), with a calculated EC 50 (half-maximal inhibitory concentration) of 1.4 µM.

As the use of the T m shift appeared to be an interesting tool for evaluating the potential binding properties of compounds, as well documented in the literature, [START_REF] Pantoliano | High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery[END_REF] it was thus employed in the screening of the limited library in the MsmegFadD32 HTS.

Screening Cascade and Compound Progression

The biochemical assay was validated for HTS by testing its reproducibility. An elaborate assay validation was performed before the throughput screening to assess the dayto-day assay variability (data not shown). A set of 200 compounds was screened on different occasions, and the calculated Z′ value [START_REF] Zhang | A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays[END_REF] was 0.7 (data not shown), thus allowing initiation of the screening campaign. For the primary screening, 10 177 compounds were screened in singlicates (at 20 µM) and, based on the 33% inhibition cutoff and stringent statistical criteria (hits outside mean + 2.5 interquartile range [IQR]*), 412 potential inhibitors were selected (Suppl. Fig. S4). These compounds were tested for the 10-point (100 µM down to 0.2 µM) CR assay, and those showing CR and EC 50 values less than 100 µM were progressed further. Of 412 compounds, 200 were shortlisted: 98 compounds with good CRs (with Hill slopes between 0.5 and 2.0) and 102 with acceptable CRs (with a relaxed criterion for Hill slopes between 0.5 and 3). These 200 CR-confirmed hits were then classified into chemical clusters. Only 32 of these compounds were further selected based on rigorous hit evaluation criteria, such as chemical properties, ligand efficiency (cutoff value = 0.3), chemical tractability, and other druglike properties. † The 32 compounds were then tested in *Compound inhibition data sets follow "normal (Gaussian) distribution," wherein the spread of the middle-half of the data is called interquartile range or IQR. Statistically, this spread is 4σ/3, where σ is the standard deviation. Statistically significant inhibition data points usually reside beyond the Median + 2 * IQR range as "outliers." These outliers deserve attention as potential inhibitors. †If the compound is identified as a hit, it should satisfy the following hit evaluation criteria: (1) chemical nature: should have drug-like properties with adequate lipophilicity, ligand efficiency, and so on; (2) chemical tractability: should have multiple modification points to be able to make several analogues to form a chemical series in order to explore the most desirable derivative; and (3) pharmacophores: should not have known toxic motifs. parallel in the promiscuity test (high protein effect) and the Thermofluor (T m shift) assays. In the promiscuity test, CR studies were done simultaneously at 1× and 5× protein concentrations to ascertain an undesirable protein effect or nonspecific inhibition. A compound is defined as "promiscuous" (i.e., exhibits undesirable protein effect) when it shows a 2-fold or more increase in its EC 50 value at higher (5× or higher) protein concentrations. The 2-fold shift criterion for determining the promiscuity was used since, for nonpromiscuous compounds, even a large change in enzyme concentration (2×-10×) is not expected to lead to changes in the EC 50 as the inhibitor's concentration is in far more excess than that of the protein, and unless the inhibitor binds nonspecifically, the EC 50 s remain within a 2-fold change. [START_REF] Feng | A Detergent-Based Assay for the Detection of Promiscuous Inhibitors[END_REF][START_REF] Mcgovern | A Common Mechanism Underlying Promiscuous Inhibitors from Virtual and High-Throughput Screening[END_REF][START_REF] Shoichet | Interpreting Steep Dose-Response Curves in Early Inhibitor Discovery[END_REF] Sixteen compounds were found to pass this test (i.e., these compounds did not show a high protein effect) (Suppl. Fig. S4).

The Thermofluor assay was done for the same 32 compounds to ascertain a significant dose-dependent shift in MsmegFadD32 T m as a result of binding of the compounds to the enzyme. Five of these compounds showed dose-dependent binding to the enzyme (Suppl. Table S1A) and did not show a nonspecific high protein effect and thus were "nonpromiscuous" in the promiscuity test (data not shown). Together, these data suggest that these compounds bind to the enzyme specifically. To substantiate these results, competition studies were done, where binding of all 16 nonpromiscuous compounds to MsmegFadD32 was checked in the presence of ATP-γ-S in the Thermofluor assay. Convincingly enough, the same five compounds indicated dose-dependent binding, preferentially to the free form of the enzyme (with ∆T m 1 > ∆T m 2), suggesting that they are competitive inhibitors of MsmegFadD32 and that they competed with the ATP analogue for binding to the enzyme (Suppl. Table S1B). Binding of any of these compounds to the enzyme in the presence of the second substrate, lauric acid, however, could not be demonstrated because compounds did not show significant T m shifts in the presence of C12. The latter alone or in the presence of ATP-γ-S did not show significant T m shifts upon binding to MsmegFadD32 (data not shown). It is not uncommon that some substrates do not show significant T m shifts upon binding to enzymes (in-house data) and that their binding to the enzyme can be shown with other biophysical assays such as 1D nuclear magnetic resonance spectroscopy. [START_REF] Meyer | NMR Spectroscopy Techniques for Screening and Identifying Ligand Binding to Protein Receptors[END_REF] These five hits represent five chemical classes as shown in Figure 4B, thus exhibiting (1) dose-dependent enzyme inhibition, (2) no protein effect (nonpromiscuous inhibitors), and (3) binding to MsmegFadD32 in the Thermofluor assay.

Thus, this screening campaign demonstrated that the essential FadD32 enzyme is a "druggable" target. This HTS was performed on the M. smegmatis FadD32 protein using a 10 000-compound limited library and has validated the optimized HTS assay, leading to the selection of five compounds as inhibitors of this enzyme with a high hit rate (4%). These are true inhibitors, as shown by binding as well as competition studies. This proof of concept being made, the perspective of this work will be to apply the same strategy using an HTS campaign with an extended library to select more compounds with potentially lower EC 50 . The selected molecules will be the starting point for testing the corresponding chemical classes through "backscreening" on the M. tuberculosis enzyme. The efficacy of the most potent molecules will be tested on M. tuberculosis growth and checked on sensitive and resistant isolates. The application of high-throughput chemical screening for the discovery of small molecules that could inhibit FadD32 activity has significant potential in the development of novel anti-TB drugs. m shift measurements of the five selected compounds. $ Competes with ATP-γ-S in a dose-dependent manner for binding to the enzyme. For details, refer to Supplementary Table S1B.

Figure 1 . 24 -C 26 )

 12426 Figure 1. The FadD32 activity, purification, and assay adaptation. (A) Role of FadD32 in the biosynthesis of mycolic acids. The synthesis of mycolic acids involves the intervention of fatty acid synthases (FAS): FAS-I for de novo synthesis and FAS-II for elongation of fatty acids (FAs). The condensation (by Pks13) between two activated fatty acids produces, after reduction (by the ketoacyl reductase CmrA), the mature mycolic ester with the characteristic mycolic motif (alpha-branch, beta-hydroxyl). Both condensation substrates are activated: The acyl-CoA (C 24 -C 26 ) is carboxylated by the acyl-CoA carboxylase complex (AccD4-containing complex), yielding the alpha-branch (R 2 ) of mycolic acids. The very long-chain fatty acid (meromycolic acid, C 50 -C 60 ) is activated as acyl-AMP by FadD32 (FAAL32), at the origin of the meromycolic chain (R 1 ) of mycolic acids. R 1 and R 2 represent long hydrocarbon chains, and X is an unknown acceptor. (B) A schematic representation of FAAL reaction catalyzed by FadD32 and detection of inorganic phosphate (Pi) using the Pyrophosphatasecoupled reaction. PPi is pyrophosphate. (C) Purity of the different FadD32 orthologues after affinity and gel filtration chromatography purification. Protein fractions were analyzed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (D) FAAL activity of FadD32 orthologues: Mycobacterium tuberculosis (dotted line), Corynebacterium glutamicum (solid line), Mycobacterium smegmatis (dashed line), and inset. Assays were conducted in Tris-HCl (pH 8) using 200 µM lauric acid (C 12 ) and 2 mM adenosine triphosphate (ATP) as substrates. Serial dilutions of enzymes were performed in Tris-HCl (pH 8). Means and standard errors of the mean (SEM) of four replicates are shown. Lines were obtained by linear regression, and data are representative of at least three independent experiments.

Figure 2 .

 2 Figure 2. Assay optimizations using the FadD32 from Mycobacterium smegmatis. (A) Melting temperature (T m ) measurements of MsmegFadD32 as a function of pH. The differential scanning fluorimetry measurements for the protein at the indicated pH are displayed. (B) Optimization of buffer and pH: Assays were conducted during 80 min in different buffers using 100 nM of the MsmegFadD32 enzyme. Inset: Fatty acyl-AMP ligase (FAAL) activity of MsmegFadD32 at different pH. Serial dilutions of enzymes were performed in the indicated HEPES buffer (dashed line: pH 6.5; dotted line: pH 7.5; solid line: pH 7.0) using 200 µM lauric acid and 2 mM adenosine triphosphate (ATP) as substrates. Means and standard errors of the mean (SEM) of four replicates are shown. Lines were obtained by linear regression. (C) Time course of MsmegFadD32 activity. Assays were conducted at room temperature in HEPES pH 7.5 using 20 µM lauric acid and 0.2 mM ATP using 80 nM (solid line), 40 nM (dashed line), or 20 nM (dotted line) of enzyme.

Figure 3 .

 3 Figure 3. Fatty acyl-AMP ligase (FAAL) activity of MsmegFadD32 after assay optimizations. (A) Radio-thin-layer chromatography (TLC) analysis of the [1-14 C]C12-AMP synthesized using increasing concentrations of the MsmegFadD32 enzyme. (B) A protein dose-response curve of MsmegFadD32 showing the formation of enzyme product using either inorganic phosphate (Pi) released in the PiColorLock-based assay (Innova Biosciences, Cambridge, UK) (open squares) or acyl-AMP formed in the radio-TLC assay (black triangles). Assays were performed at room temperature during 80 min in HEPES pH 7.5 using 40 µM lauric acid and 2 mM adenosine triphosphate (ATP) as substrates. Means and standard errors of the mean (SEM) of four replicates (PiColorLock assay) or duplicates (radio-TLC) are shown. Lines were obtained by linear regression, and data are representative of three independent experiments.

Figure 4 .

 4 Figure 4. Inhibition of MsmegFadD32 by the substrate analogue AMPC12 and screening for inhibitors of its activity. (A) Inhibition curve of MsmegFadD32 activity by AMPC12. The chemical structure of adenosine 5′-alkylphosphate (alkyl-AMP) is displayed; R = C12 for AMPC12. Increasing concentrations of the compound were added to the assay, and FadD32 activity was measured in HEPES pH 7.5 in the presence of 20 µM lauric acid (C 12 ) and 0.2 mM adenosine triphosphate (ATP) as substrates. Reactions were conducted at room temperature during 80 min and were done in quadruplicates. (B) Chemical classes, EC 50 ,and Tm shift measurements of the five selected compounds. $ Competes with ATP-γ-S in a dose-dependent manner for binding to the enzyme. For details, refer to Supplementary TableS1B.

Table 1 .

 1 Properties of the Selected FadD32 Orthologues from Mycobacterium Species and Corynebacterium glutamicum

			Purification	Specific Activity (200 µM Lauric	Volume of Culture for a
	FadD32 Protein	Identity, %	Yield, mg/L	Acid), nmol•min -1 •mg -1	100-Microplate Assay, L
	M. tuberculosis	100	7	32 ± 2	14
	M. smegmatis	75	45	2443 ± 99	0.08
	C. glutamicum	40	50	71 ± 2	1

Table 2 .

 2 Apparent Kinetic Parameters of MsmegFadD32

	Substrate	K m , µM	V max , pmol•min -1	k cat , min -1	k cat /K m	, min -1 µM -1
	ATP	76.0 ± 6.7	16.8 ± 0.4	14.0 ± 0.3		0.18
	C10	259.0 ± 57.9	19 ± 2.8	16.1 ± 2.4		0.06
	C12	25.9 ± 1.6	13.5 ± 0.3	11.2 ± 0.2		0.43
	C14	5.2 ± 0.5	5.0 ± 0.1	4.2 ± 0.1		0.81
	Initial velocities of the FadD32 reaction were assayed spectrophotometrically using the coupled assay. Reactions were conducted at room temperature
	during 40 min in HEPES pH 7.5 as described in Materials and Methods. The steady-state apparent parameters K m (Michaelis constant), V max (maximum velocity), k cat (catalytic constant), and k cat /K m (catalytic efficacy) were determined using nonlinear regression to fit the data to the Michaelis-Menten equation. ATP, adenosine triphosphate.

at BIBLIOTHEQUE INTERUNIV SANTE on January 30, 2013 jbx.sagepub.com Downloaded from

Acknowledgments

We are grateful to Dr. Christian Chalut (IPBS, Toulouse) for providing the pET15b-MtubfadD32 clone. Special thanks to Prashanti Madhavapeddi (AstraZeneca Infection iMed) for her inputs on the Thermofluor assay. We would also like to thank Adam Shapiro and Suresh Solapure (AstraZeneca Infection iMed) for critically reviewing the manuscript. The DSF equipment used in this study is part of the Integrated Screening Platform of Toulouse (PICT, IBiSA).

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 Centre National de la Recherche Scientifique (France), the MYCA contract from the Région Midi-Pyrénées and FEDER Funds from the European Community that funded the postdoctoral fellowships of S.G., and the European Community New Medicines for Tuberculosis (grant LSHP-CT-2005-018923).

Declaration of Conflicting Interests

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

A B

Supplemental Material

Suppl. Fig. S1: Michaelis-Menten curves for the determination of the appatent K m and V max for lauric acid (C 12 ) and ATP. Data were obtained using the PiColorLock-based assay.

Reactions were conducted at RT during 40 min in HEPES pH7.5 buffer using increasing concentration of lauric acid with 2mM ATP (A) or increasing concentration of ATP with 200 µM of lauric acid (B). The K m and V max were determined using non-linear regression to fit the data to the Michaelis-Menten equation.


Sher Ahmad

Wassim Sebai

Marie-Pierre Belleville

Nicolas Brun

Anne Galarneau

J Sanchez-Marcano
email: jose.sanchez-marcano@umontpellier.fr

ENZYMATIC DEGRADATION OF MICROPOLLUTANTS IN WATER: THE CASE OF TETRACYCLINE DEGRADATION BY ENZYMES IMMOBILIZED ON MONOLITHS

Enzymatic monolithic reactors were applied for the degradation of micropollutants through flow-through reactor configuration. Silica monoliths with uniform macro-/mesoporous structures (20 µm and 20 nm macroand mesopores diameters) high porosity (83%) and high surface area (370 m2 g-1) were prepared. The monoliths were cladded in steel tubing and laccase from Trametes versicolor was immobilized by covalent grafting. Enzymatic monoliths presented a very good oxidation activity and were used for the degradation of tetracycline (TC) in aqueous solutions in a tubular plug reactor with recycling configuration. TC degradation efficiency was found to be 40-50 % after 5 h of reaction at pH 7. The immobilized laccase on silica monoliths exhibited high operational stability during 75 hours of sequential operation.

Introduction

Pharmaceuticals are currently used in human and animal medicine. Many of these molecules or their metabolites are currently discharged in wastewater but they are not completely removed by conventional wastewater treatment plants. Indeed, they end up in surface and underground waters (U. [START_REF] Szymańska | Presence of antibiotics in the aquatic environment in Europe and their analytical monitoring: Recent trends and perspectives[END_REF], [START_REF] Halling-Sørensen | Toxicity of Tetracyclines and Tetracycline Degradation Products to Environmentally Relevant Bacteria, Including Selected Tetracycline-Resistant Bacteria[END_REF], [START_REF] Watkinson | Removal of antibiotics in conventional and advanced wastewater treatment: Implications for environmental discharge and wastewater recycling[END_REF]). Among these molecules, antibiotics are a special family of pharmaceutical molecules that are causing increasingly frequent antibiotic resistance in patients. They have therefore become a real public health problem. Antibiotics, like tetracycline (TC) are currently found in wastewaters and natural water bodies increasing the ecotoxicity and anti-microbial resistance [START_REF] Halling-Sørensen | Toxicity of Tetracyclines and Tetracycline Degradation Products to Environmentally Relevant Bacteria, Including Selected Tetracycline-Resistant Bacteria[END_REF]). There is a real need to develop alternative technologies techniques for the complete and efficient removal of these pharmaceuticals. Biodegradation of antibiotics is recently been explored using oxidoreductases enzymes as a bio-catalyst [START_REF] Varga | Enzymatic treatment and subsequent toxicity of organic micropollutants using oxidoreductases -A review[END_REF]. Laccase belongs to a group of polyphenol oxidases containing copper atoms in the catalytic center and usually called multicopper oxidases.

In contrast to peroxidases, laccase does not require the presence of hydrogen peroxide or manganese for oxidation and needs only oxygen as the final electron receptor offering a green alternative in wastewater treatment [START_REF] Primozic | Immobilized Laccase for Sustainable Technological Processes[END_REF]. Due to its biochemical structure, Laccase has ability to degrade large variety of micro pollutants at ambient temperatures and can be considered as good green catalysts for degradation of micro pollutants [START_REF] Kües | Fungal enzymes for environmental management[END_REF]. Immobilizing laccases on different solid supports like membranes have been tested for TC degradation and resulted being more active than free laccases while showing a good stability [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF]. However, the degradation rates reported are limited by the small surface area available in membranes for enzymes immobilization.

Enzymatic monoliths are interesting candidates to be applied in continuous flow through plug reactors. In this operating mode, the flow is forced to pass exclusively through the internal monolith porosity where the biocatalyst is immobilized, intensifying the probability of contact between the reactants and the enzyme during the mass transfer process. Indeed, this configuration results in improved control of the reaction through the "micro-reactor concept" (Westermann, 2009).

Silica monoliths featuring large surface area (400 -700 m2/g), porosity and permeability have been successfully used for process intensification in catalytic reactions under continuous flow [START_REF] Galarneau | Hierarchical porous silica monoliths: A novel class of microreactors for process intensification in catalysis and adsorption[END_REF]. Herein in this work we show that these new kind of monoliths are also promising supports to immobilize enzymes, as laccases, for improving the degradation rate of pollutants like TC. In this work silica monoliths with hierarchical porosity (macro-/mesoporosity) were prepared by a controlled sol-gel process and a Laccase form (trametes versicolor) has been grafted on the internal surface of the monoliths. Then, activated monoliths were immediately used for TC degradation in a plug flow reactor (PFR) with continuous recycling. Finally a model coupling the enzymatic kinetics with mass transfer was built in order to optimize the enzymatic reactor.

Experimental

Materials

Polyethylene glycol (PEG) (99%, 100 kDa), tetraethoxysilane (TEOS) (99%), (3-aminopropyl) triethoxysilane (APTES) (99%), commercial powder of laccase from Trametes versicolor (activity ≥0.5 U mg-1), tetracycline (TC) (≥98.0%), glutaraldehyde (25% v/v) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (≥98.0%,) were purchased from Sigma-Aldrich.

Silica monoliths synthesis, functionalization and characterization

Silica monoliths with hierarchical porosity (macro-/mesoporosity) were prepared by a controlled sol-gel process with TEOS and PEG [START_REF] Galarneau | Hierarchical porous silica monoliths: A novel class of microreactors for process intensification in catalysis and adsorption[END_REF]. Prior to enzyme immobilization, the monoliths were preactivated by grafting amino groups with APTES in ethanol under reflux (80 °C) overnight. Tubular reactors were built by cladding the monoliths inside Teflon™ heat shrinkable gains at 180 °C for 2 h to be finally connected to stainless-steel tubing. The enzyme grafting of monoliths was carried out by a successive immersion of the pre-activated monoliths in a first glutaraldehyde solution (4 % v/v), a washing step and a second commercial laccase (Trametes versicolor) solution (5±1 U/mL), both solutions were prepared in a citrate phosphate buffer (pH 7, 0.1 M), the same buffer was used for washing. After a last washing the activated monoliths were immediately used for the determination of activity (ABTS) in a stirred tank reactor or TC degradation in a plug flow reactor (PFR) with continuous recycling.

Monoliths were characterized structurally by scanning electron microscopy (SEM) (Hitachi S-4800 I FEG-SEM). Porosity was determined by mercury porosimetry (Micrometrics Autopore 9220) and nitrogen adsorption at 77 K (Micromeritics Tristar 3020). The quantitative determination of the number of amine functions on the surface of the silica monolith was determined by TGA analysis (PerkinElmer STA 6000).

Determination of immobilized enzymes activity

The activity (U/mgmonolith) was reassured using freshly activated monoliths which were gently crushed and a portion of the activated powder was used for the oxidation of ABTS (1mM) prepared in a citrate phosphate buffer at pH 4 on a stirred tank reactor. The activity was measured by the change in absorbance of an standard ABTS solution at 420nm (UV-VIS 2401 Shimadzu spectrometer). One unit of activity (U) corresponds to quantity of enzyme required for the oxidation of 1µmol of ABTS per one minute. The immobilization yield was calculated taking into account the activity of the enzymatic solution used for immobilization before and after activation process.

Determination of reaction kinetics parameters

Reaction kinetics parameters (KM and VMAX) of ABTS oxidation by free and immobilized laccase were measured with a solution of ABTS (20-100µM) at 25°C in a citrate phosphate buffer 0.1M (pH 4) by the spectrometric methodology described above. Lineweaver-Burk plot was used to estimate kinetic parameters, KM and Vmax according to the following equation:

1 𝑉 = 𝐾 𝑀 𝑉 𝑚𝑎𝑥 1 [𝑆] + 1 𝑉 𝑚𝑎𝑥 (1)
Where [S] is the substrate concentration (µmol/L), V and VMAX are reactively the reaction rate and maximum reaction rate (µmol/min) and KM the Michaelis constant (µmol/L).

Tetracycline degradation experiments

TC degradation tests were carried out with three laccase-activated monoliths connected in series (0.6 cm diameter and 0.5 cm length, each, equivalent to a total of 15.0 U ± 0.5 U of enzymatic activity). TC solution (20 mg/L) prepared in osmosed water pH 6, at 25°C was continuously flowed through the enzymatic monoliths (HPLC pump, Gibson model: 321, France) and constantly recycled from a reservoir. The flow rate was kept at 1 mL/min. These experiments were carried out also to determine the stability of laccase-activated monoliths under continuous operation for TC degradation. Indeed, degradation tests were carried out following a sequential procedure: after ~20 hours of reaction the pilot was emptied, rinsed with osmosed water and kept at room temperature overnight, then a new fresh TC solution was introduced and the run start again. The cycles were repeated for a total duration of 75 hours. Samples were taken from the reservoir and analysed by HPLC coupled to triple-quadrupole mass spectrometry (HPLC-MS). Samples were injected through a Macherey-Nagel C18 column (50 mm x 2 mm) with a Waters e2695 Separations Module, and the 410 m/z fragment was detected with a Micromass Quattro micro API device.

Results

Characterization of monoliths

The monoliths were characterized by SEM (Figure 1), mercury porosimetry, nitrogen adsorption at 77 K and thermogravimetric analysis. The monoliths presented homogeneous and interconnected flow-through macropores of 20~25 μm diameter, mesopores diameter of 20 nm, present a very high porosity (Vmacro = 3.4 mL g -1 , Vmeso = 0.8 mL g -1 , total porosity ε = 0.83) and a large surface area (370 m 2 g -1 ). The thermogravimetric analysis allowed calculating the amount of amino functions of pre-activated monoliths by the loss of weight between 200°C to 900°C (1 mmol NH2 g monolith -1 ). After laccase immobilization some structural values changed. We observed a decrease of the specific surface (185 m2 g -1 ), Vmeso (0.4 mL g -1

)

and mesopores diameter (14 nm).

Immobilization of enzymes and activity measurement

The measured activity of monoliths powder was 0.18 U/mgmonolith. The immobilization yield was equal to 80%, which was much higher than the immobilization yield observed in case of membranes as find by [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF]. This very high immobilization yield confirms that monoliths can graft large amount of active enzymes. The activity of a single monolith (6 mm diameter and 5 mm length, mass of 50 mg) was calculated from the measured activity of crushed monoliths explained above. The mean activity of a single monolith was of 7.8 ± 0.5 U.

Determination of reaction kinetic parameters

Enzymes reaction kinetic parameters (KM and VMAX) are important factors to be measured because they describe the activity of enzymes towards a substrate conversion. Kinetic parameters for both free and immobilized enzymes were determined by the method as discussed above. Measured values are shown in Table1. It can be observed that for the same concentration of free and immobilized enzymes: (0.0085 U/mL) VMAX is a little bit higher for immobilized enzymes. Likewise, KM is lower for free enzymes. This result indicates that immobilized has a higher activity and a lower affinity towards the substrate than free enzyme. This result is surprising because generally structural conformation and diffusion limitations occur after immobilization [START_REF] Salami | Covalent immobilization of laccase by one pot three component reaction and its application in the decolorization of textile dyes[END_REF]. However, in some cases immobilized enzymes has resulted on VMAX enhancement as reported for laccase immobilized on membranes [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF], or lipase immobilized on silica [START_REF] Kumar | SiO2 microparticles with carbon nanotube-derived mesopores as an efficient support for enzyme immobilization[END_REF].

Table 1: Reaction kinetic parameters for the oxidation of ABTS with free and immobilized laccase. This result can be explained by the decrease of substrate concentration, if the kinetics obeys to a Michaelis-Menten equation. However, the decrease of the oxygen content, or even the formation of inhibiting byproducts can also be considered. [START_REF] Kurniawati | Stoichiometry during the laccase-catalysed oxidation of aqueous phenol[END_REF] and [START_REF] Ortner | Laccase mediated oxidation of industrial lignins: Is oxygen limiting?[END_REF], have noticed that the velocity of oxidation of substrates by laccases can decrease very rapidly during the first hours of reaction because the oxygen concentration reduction near catalytic sites. Indeed, the conversion can be improved by stirring on air atmosphere or reach 100% by using pure oxygen [START_REF] Ortner | Laccase mediated oxidation of industrial lignins: Is oxygen limiting?[END_REF]). In the case of experiments carried out in the monolithic system no stirring under air or oxygen was done. Then, it is conceivable that oxygen depletion nearby catalytic sites caused the degradation rate decrease.

The operational stability of enzymatic monoliths was studied during 75 hours with the sequential operation procedure described in section 2.5; the results are displayed in Figure 3. We can notice that the evolution of TC concentration is very similar for all of the cycles. As it was discussed above, TC concentration decreases very quickly during the first hours of reaction and then the degradation rate go-slow. When the solution is changed with a fresh one containing the initial TC and oxygen concentration the same pattern of TC reduction is obtained. Indeed we can conclude that the enzyme immobilized in monoliths is stable during at least 75 hours of sequential operation.

Conclusions

The preliminary results presented in this work show that the laccase immobilization in silica monoliths results is very promising for tetracycline degradation. Silica monoliths with a homogenous mesoporous/macroporous structure featuring, high specific surface area and porosity were successfully prepared. These monoliths were pre-activated with APTES and glutaraldehyde in order to covalently immobilize laccase from Trametes versicolor. The activity and kinetic parameters were determined for ABTS as model molecule. From reaction kinetic parameters VMAX and KM values, it was concluded that after immobilization laccase was a little bit more active but presents lower affinity towards the substrate. Enzymatic monoliths were successfully implemented at laboratory scale for the degradation of tetracycline as model micropollutant in a continuous plug flow mode with recycling. Indeed, approximately 40-50% of TC in 20 ppm solutions was degraded in 5 hours. The immobilized laccase on silica monoliths exhibited high operational stability during 75 hours of sequential operation this factor is fundamental for the applicability of such enzymatic reactors at large scale.

Figure 1 :

 1 Figure 1: SEM of monoliths showing uniform macroporous structure.

3. 3

 3 Tetracycline degradation experiments TC (20 ppm) degradation results are displayed in Figure 2. Control experiments (blank tests) were carried out to study the effect of TC self-degradation or adsorption on the evolution of TC concentration. Blank experiments were carried out with enzymatic monoliths thermally deactivated by heating in oven at 100°C for 2 hours. The results of blank tests show that less than 2% of initial TC was depleted. The evolution of TC concentration (percentage of the concentration/initial concentration) shows that laccase-activated monoliths were able to deplete 40% of initial TC during the first 5 hours and then the degradation rate decreases.

Figure 2 :

 2 Figure 2: Evolution of % TC concentration (concentration/initial concentration).

Figure 3 :

 3 Figure 3: Evolution of TC degradation during 75 hours of sequential operation. Red lines indicate the time when the pilot was emptied, rinsed with osmosed water and filled again with a new fresh TC solution (20 mg/L).

Acknowledgments

This research was funded by the ANR French agency, project MUSE ANR-16-IDEX-0006 project DEMEMO.

Mr. S. Ahmad acknowledges the Higher Education Commission, Pakistan for the PhD scholarship.