Mn(III) Pyrophosphate as an Efficient Tool for Studying the Mode of Action of Isoniazid on the InhA Protein of Mycobacterium tuberculosis

The antituberculosis drug isoniazid (INH) is quickly oxidizedby stoichiometric amounts of manganese(III) pyrophosphate. Inthe presence of nicotinamide coenzymes (NAD⁺, NADH, nicotinamidemononucleotide [NMN⁺]) and nicotinic acid adenine dinucleotide(DNAD⁺), INH oxidation produced the formation of INH-coenzymeadducts in addition to known biologically inactive products(isonicotinic acid, isonicotinamide, and isonicotinaldehyde).A pool of INH-NAD(H) adducts preformed in solution allowed therapid and strong inhibition of in vitro activity of the enoyl-acylcarrier protein reductase InhA, an INH target in the biosyntheticpathway of mycolic acids: the inhibition was 90 or 60% whenthe adducts were formed in the presence of NAD⁺ or NADH, respectively.Under similar conditions, no inhibitory activity of INH-NMN(H)and INH-DNAD(H) adducts was detected. When an isolated poolof 100 nM INH-NAD(H) adducts was first incubated with InhA,the enzyme activity was inhibited by 80%; when present in excess,both NADH and decenoyl-coenzyme A are able to prevent this phenomenon.InhA inhibition by several types of INH-coenzyme adducts coexistingin solution is discussed in relation with the structure of thecoenzyme, the stereochemistry of the adducts, and their existenceas both open and cyclic forms. Thus, manganese(III) pyrophosphateappears to be an efficient and convenient alternative oxidantto mimic the activity of the Mycobacterium tuberculosis KatGcatalase-peroxidase and will be useful for further mechanisticstudies of INH activation and for structural investigationsof reactive INH species in order to promote the design of newinhibitors of InhA as potential antituberculous drugs.

INTRODUCTION

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Introduction

Tuberculosis, an infectious disease caused by Mycobacteriumtuberculosis, remains one of the leading causes of death inthe world. Isoniazid (INH), a frontline antibiotic used in thetreatment of tuberculosis (3, 4), is considered to be a prodrugthat requires activation by the M. tuberculosis catalase-peroxidaseKatG (10, 28). However, none of the stable derivatives observedin KatG-dependent INH conversion, i.e., isonicotinic acid (product1), isonicotinamide (product 2) and isonicotinaldehyde (product3) (Fig. 1) have demonstrated a bactericidal effect (9). Studies(22, 23, 26) have suggested that the activated form of INH,probably an isonicotinoyl radical, is capable of reacting withthe ß-NAD (NAD⁺/NADH) which is the cofactor of thelong-chain 2-trans-enoyl-acyl carrier protein reductase InhA(2, 20). InhA is a key enzyme involved in the biosynthesis oflong-chain fatty acids and of mycolic acids, specific componentsof the mycobacterial cell wall (5, 15). The formation of thecovalent adduct(s) INH-NAD(H) as a competitive inhibitor(s)might explain the loss of InhA activity and interruption ofmycolic acid synthesis (20, 22). Despite recent efforts, theexact nature of the active form of INH and the precise activationmechanism of this drug are still a matter of debate. Severalstudies have been focused on the role of the KatG protein (8,10, 14, 25, 28), a hemoprotein having a manganese-dependentperoxidase activity (13, 27). KatG catalyzes the conversionof Mn(II) to Mn(III) (13), the latter metal ion being probablyable to oxidize INH (this mode of activation is reminiscentof the manganese peroxidase mechanism of the white-rot fungusPhanerochaete chrysosporium [24]). In addition, since Mn(II)/O₂is a poor activating system (7, 12), INH activation has beendirectly performed with Mn(III) salts (7) and was shown to giveoxidation products 1 and 2 (13). The possibility of using Mn(III)to oxidize INH and form InhA inhibitors has been briefly mentioned,but without any experimental details (26).

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FIG. 1. Structures of INH, stable oxidation products 1 to 3, and proposed structures for INH-NAD(H) or INH-DNAD(H) adducts observed in solution.

In the present work, we investigate the activation of INH bystoichiometric amounts of Mn(III) pyrophosphate, a stable formof Mn(III) ions in aqueous solutions previously used in ourmodel studies of the manganese peroxidase of P. chrysosporium(6). Since Mn(III) is a strong oxidant which undergoes spontaneousdismutation in Mn(II) and Mn(IV) in water, we chose pyrophosphateas an oxidant-resistant chelating agent to stabilize Mn(III)in the pH range from 4 to 6. Other organic chelating agentssuch as malate, malonate, lactate, oxalate, or tartrate arenot as stable over time and show storage problems. In a previouswork (18), we demonstrated that a stoichiometric amount of Mn(III)pyrophosphate can replace either the use of Mn(II)/O₂ or thecatalysis by the KatG protein in the activation of INH. Formationof a series of adducts was detected and shown to be the resultof acylation in position 4 of the nicotinoyl moiety of the coenzymeby the isonicotinoyl radical generated from INH (with creationof a new chiral center at position 4 and therefore formationof two epimeric adducts; see structures 4 and 5 or 11 and 12in Fig. 1, for INH adducts with NAD⁺ and nicotinic acid adeninedinucleotide [DNAD⁺], respectively). An additional spontaneouscyclization process creates a second chiral center at position7, which gives four new diastereoisomeric compounds having ahemiamidal structure (see structures 6 to 9 in Fig. 1). Thecoexistence in solution of these six dihydropyridine derivatives(two open and four cyclized) was clearly demonstrated for NAD⁺(17). A typical high-performance liquid chromatography (HPLC)profile is shown in Fig. 2A. In the case of INH-DNAD adducts,the carboxylic group of the nicotinic moiety (instead of theamide group present in NAD⁺) does not allow the cyclizationprocess, and only the two open structures, compounds 11 and12, were observed (Fig. 2B). It should be noted that a smallamount of oxidized adducts (the dihydropyridine ring being convertedinto a pyridinium ring) can also be detected (peak 10 in Fig.2A, peak 13 in Fig. 2B, and compounds 10 and 13 in Fig. 1).In the case of reaction with NAD⁺, the two main adducts, compounds6 and 7, analyzed by liquid chromatography-electrospray ionization-massspectrometry (LC-ESI-MS), showed a molecular weight (770.1)identical and UV characteristics ( ${lambda}$ _max = 260 and 330 nm, ${varepsilon}$ ₂₆₀/ ${varepsilon}$ ₃₃₀= 3.2 to 3.5) similar to those reported for the adduct recentlyisolated after activation of INH in the presence of KatG andInhA (11). All these data are also consistent with the proposedformula of the bound inhibitor present in the crystal structureof the InhA-inhibitor complex (Fig. 3) (22), making it the mostlikely inhibitor of InhA.

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FIG. 2. HPLC profiles of reaction mixtures containing INH (2 mM), NAD⁺ (2 mM) (A) or DNAD⁺ (2 mM) (B), and Mn(III) pyrophosphate (4 mM; introduced in 10 consecutive additions of 400 µM each, every 2 min) in phosphate buffer (100 mM), pH 7.5. See Fig. 1 for the compound formula corresponding to each peak. Compounds 4 and 10 coelute and compound 4 is the minor component. _* and _*_*, nicotinamide and nicotinic acid coming from decomposition of NAD⁺ and DNAD⁺, respectively. RRT, relative retention time (with INH as reference).

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FIG. 3. Structure of the INH-NAD(H) adduct within the active site of InhA as proposed on the basis of X-ray crystallography (22).

However, several questions remained; for instance, are adductsgenerated by oxidation in the presence of the chemical activatorMn(III) pyrophosphate able to inhibit InhA, and what is theincidence of the cyclization process observed for INH-NAD(H)adducts on their potential inhibitory activity? In the presentwork, we investigate (i) the ability of Mn(III) pyrophosphateto oxidize INH in the presence of various nicotinic coenzymeswith formation of INH adducts and (ii) the potency of theseadducts to inhibit InhA.

MATERIALS AND METHODS

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Materials and Methods

Analytical methods. (i) HPLC analyses. HPLC analyses were performed on a reverse-phase C₁₈ column (particlesize, 10 µm; 250 by 4.6 mm; Nucleosil) using a 5:95 (vol/vol)methanol-5 mM ammonium acetate (NH₄OAc) (pH 4.5) solution aseluent (Table 1, runs 1 and 2) or a linear gradient from 0 to100% acetonitrile in 70 mM NH₄OAc aqueous solution for INH-coenzymeadduct {INH-NAD(H), INH-DNAD(H), INH-nicotinamide mononucleotide[NMN(H)] adducts} (Table 1, runs 3 to 6) analyses (flow rate:1 ml min^-1). The column was coupled to a diode array detector(Kontron) for the detection of products at 260 nm and the monitoringof UV-visible spectra. Yields were calculated for INH and compounds1 to 3 by comparison with authentic sample calibration curves(in order to detect compound 3 as its O-carboxymethyl oximederivative, some reaction samples were analyzed after 5 minof quenching with 20 mM NH₂OCH₂COOH) and, for INH adducts, byusing the same ${varepsilon}$ ₂₆₀ values as for corresponding coenzymes. Collectionof HPLC-separated peaks was performed on a reverse-phase C₈semipreparative column (particle size, 10 µm; 250 by 10mm; Nucleosil) using detection at 260 and 330 nm (this lastmaximum absorbance was used as an indicator for dihydropyridineadduct formation). Elution conditions are precisely describedunder "Purification of INH-coenzyme adducts" below (flow rate,2.5 ml min^-1).

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TABLE 1. Determination of INH derivatives produced after nonenzymatic activation in the absence or presence of nicotinamide or nicotinic coenzymes

LC-ESI-MS analyses . LC separations were performed under the conditions indicatedabove but with a quaternary pump. Only 4% of the flow elutedfrom the column was introduced into the electrospray sourceafter automated mixing with a 1% HCOOH solution using a HarvardApparatus syringe pump. The ESI-MS spectrometer was a Perkin-ElmerSCIEX API 365, and analyses were performed in the positive mode.

All InhA assays were performed on a Uvikon 923 spectrophotometer(Kontron) equipped with a circulating water bath for temperaturecontrol.

(ii) Materials. Manganese(III) pyrophosphate, [Mn^III(H₂P₂O₇)₃]Na₃, was preparedaccording to the method of Archibald and Fridovich (1) and followingthe improved protocol previously described (17). M. tuberculosisInhA was overexpressed in Escherichia coli and purified as previouslydescribed (21). INH, nicotinamide coenzymes, and analogs wereobtained from Sigma-Aldrich. The substrate 2-trans-decenoyl-coenzymeA (-CoA) was synthesized from 2-trans-decenoic acid using themixed anhydride method and purified according to the proceduredescribed elsewhere (19, 21). The concentration of the substratewas determined on the basis of ${varepsilon}$ ₂₆₀ being equal to 16,800 M^-1cm^-1.

INH oxidation. Run 1 (Table 1) involved oxidation with Mn(III) pyrophosphate;the reaction medium (final volume, 1 ml), which contained 100mM phosphate buffer (pH 7.5), 500 µM INH, and 1 mM Mn(III)pyrophosphate, was stirred at room temperature (RT) for 10 minand then analyzed by HPLC after 5 min of quenching with 20 mMNH₂OCH₂COOH (200 µM 2-nitrobenzoic acid was used as theinternal standard). The corresponding control reaction withoutoxidant (run 2) was incubated under the same conditions for48 h.

INH-coenzyme adduct formation. For runs 3 to 6 (Table 1), the reaction medium (final volume,1 ml) contained 100 mM phosphate buffer (pH 7.5), 2 mM INH,2 mM coenzyme (NAD⁺ in run 3, NADH in run 4, NMN⁺ in run 5,DNAD⁺ in run 6), and 4 mM Mn(III) pyrophosphate (introducedin 10 consecutive additions of 400 µM each, every 2 min).The reaction mixture was stirred at RT for 20 min after thelast ingredient's addition. HPLC (detection at 260 nm) and LC-ESI-MSanalyses were directly performed on the reaction mixture.

Adduct formation in the presence of InhA and simultaneous inhibition of InhA. The standard incubation medium (total volume, 50 µl) consistedof 3 µM InhA, 100 µM INH, 100 µM NADH, and200 µM Mn(III) pyrophosphate, in 100 mM sodium phosphatebuffer (pH 7.5), at 25°C (Table 2, run 1). The additionof Mn(III) pyrophosphate initiated the reaction. Control reactionswere carried out under the same conditions without Mn(III) pyrophosphate(run 2), without NADH (run 3), without INH, or without bothMn(III) pyrophosphate and INH (data not shown for the last twocontrols). Reaction aliquots (10 µl) were taken at definedtimes as specified on the x axis of Fig. 4 and analyzed forInhA activity using 2-trans-decenoyl-CoA (50 µM) as thesubstrate and NADH (100 µM) as the cofactor in 100 mMphosphate buffer, pH 7.5, at 25°C (total volume, 1 ml).In order to simplify the graphic, all control curves (exceptthat of control without NADH) are included in the hatched zoneplotted on Fig. 4.

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TABLE 2. Inhibition of InhA activity by adducts formed in the presence of InhA or by preformed adducts (INH, 100 µM; InhA, 3 µM)

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FIG. 4. (A) Inhibition of InhA activity by adducts formed in the presence of InhA. The enzyme (3 µM) was incubated in 100 mM sodium phosphate buffer, pH 7.5, at 25°C, including 100 µM INH, 100 µM NADH, and 200 µM Mn(III) pyrophosphate ( ${blacktriangleup}$ ) or no NADH ( ${square}$ ). (B) Inhibition of InhA activity by preformed adducts. Adducts were first formed by preincubation for 18 min in 100 mM sodium phosphate buffer, pH 7.5, at 25°C, containing 100 µM INH and 200 µM Mn(III) pyrophosphate and 100 µM NADH ( ${blacktriangleup}$ ), 100 µM NAD⁺ ( ${triangleup}$ , ${blacksquare}$ , and ${circ}$ ) ( ${triangleup}$ and ${blacksquare}$ , only 2 and 6 min of preincubation time, respectively), 100 µM NMN⁺ ( ${square}$ ), or 100 µM DNAD⁺ (•). Values of InhA activity obtained for each time point of a curve are expressed as a percentage of the initial enzymatic activity. Conditions are described in Materials and Methods. Hatched zones (control incubations), no INH and no Mn(III) pyrophosphate, no INH, no Mn(III) pyrophosphate.

Inhibition of InhA by a reaction mixture of preformed adducts. The preincubation reactions were performed in 50 µl (totalvolume) of 100 mM sodium phosphate buffer solution, pH 7.5,at 25°C containing 100 µM INH, 100 µM coenzyme(Table 2, NADH in run 4; NAD⁺ in run 7; NMN⁺ in run 11, or DNAD⁺in run 13), and 200 µM Mn(III) pyrophosphate. After 2and 6 min of preincubation (only for NAD⁺; runs 9 and 8, respectively)or after 18 min (all types of coenzyme), the addition of 3 µMInhA initiated the incubation reaction. Control incubation reactionswere carried out under the same conditions as those describedabove without any coenzyme (run 5), without Mn(III) pyrophosphate(run 6 for NADH; run 10 for NAD⁺; run 12 for NMN⁺, and run 14for DNAD⁺), without INH, or without both Mn(III) pyrophosphateand INH (data not shown). Reaction aliquots (10 µl) weretaken at defined times as specified on the x axis of Fig. 4and analyzed for InhA activity as described above.

Purification of INH-coenzyme adducts. The formation of INH-coenzyme adducts was performed as describedin Table 1, run 3 (pool of adducts 5 to 10 and isolated adducts6 and 7) and run 6 (isolated adducts 11 and 12). The pool ofadducts 5 to 10 was separated from other components of the reactionmixture (INH, NAD⁺, ADP-ribose, isonicotinic acid, isonicotinamide,nicotinamide, and buffer) by first washing the column for 80min with a 70 mM NH₄OAc aqueous solution and then by elutingthe pool of adducts with water and collection. HPLC reinjectionof the pool of INH-NAD(H) adducts 5 to 10 displays an HPLC profilesimilar to the adduct profile observed in Fig. 2A (peaks 5 to10). The two major peaks, peaks 6 and 7, attributed to cyclicINH-NAD(H) adducts, were purified using a linear gradient from0 to 100% acetonitrile in 70 mM NH₄OAc aqueous solution. Thepeaks 11 and 12, corresponding to INH-DNAD(H) isomeric adducts,were purified using aqueous 17 mM NH₄OAc as eluent. The chromatographicpurity of each collected INH-coenzyme adduct was estimated tobe >95%, based on subsequent HPLC reinjection. An alternativepurification process of the pool of adducts consisted of passingthe reaction mixture through a Sep-pak C₁₈ cartridge and washingwith 4 mM NH₄OAc aqueous solution. Further careful washing withwater followed by elution with acetonitrile and concentrationunder vacuum afforded the pool of adducts. After dilution inwater, HPLC reinjection of this pool of INH-NAD(H) adducts 5to 10 yielded an HPLC profile similar to the adduct profileobserved for the crude reaction mixture.

The concentration of the pool of INH-coenzyme adducts or ofisolated adducts collected either from HPLC or the Sep-pak cartridgewas estimated by UV-visible spectrum analyses using a ${varepsilon}$ ₂₆₀ of27,000 M^-1 cm^-1 and a ${varepsilon}$ ₃₂₆ of 6,900 M^-1 cm^-1 from data in theliterature (11). The pool of INH-NAD(H) adducts 5 to 10 exhibitedtwo absorption peaks at 260 nm and 326 to 328 nm and an A₂₆₀/A_326-328ratio of 3.51. The isolated INH-NAD(H) adducts 6 and 7 showedtwo absorption peaks at 260 and 328 nm and an A₂₆₀/A_326-328ratio of 3.30 (6) and 3.55 (7). Concerning the INH-DNAD(H) adducts,each fraction collected showed an absorption peak at 260 nmand a shoulder around 302 nm and an A₂₆₀/A_300-302 ratio of 3.55(11) and 3.49 (12).

Inhibition of InhA by an isolated pool of adducts and by collected single adducts. The HPLC-collected fractions containing the pool of peaks 5to 10 or one of peaks 6, 7, 11, or 12, after determination ofthe concentration, were used in InhA assays. Reactions wereinitiated by adding 60 nM InhA into a solution (total volume,1 ml) of 100 mM sodium phosphate buffer, pH 7.5, at 25°Ccontaining 100 µM NADH, 50 µM 2-trans-decenoyl-CoA,and adequate concentrations of the pool or of single collectedadducts as specified in Table 3 and, for the pool of adducts,on the x axis of Fig. 5. Control InhA assays were carried outunder the same conditions as described above using the fractionscollected at the same retention time as for the correspondingadducts, but in this case, the preincubations were performedwithout oxidant. Protection of InhA activity by the cofactoror the substrate (Fig. 6) was tested as follows. A 77 nM concentrationof InhA was preincubated for 5 min at 25°C alone (column1) or in the presence of (i) a 100 nM pool of adducts purifiedby Sep-pak (see above) (column 2), (ii) 100 µM NADH (column3), or (iii) 100 µM 2-trans-decenoyl-CoA (column 4), in100 mM sodium phosphate buffer (pH 7.5) in a total volume of800 µl. Reactions were initiated by adding 100 µMdecenoyl-CoA and 100 µM NADH (columns 1 and 2), a 100nM concentration of the purified pool of adducts and 100 µM2-trans-decenoyl-CoA (column 3), or a 100 nM concentration ofthe purified pool of adducts and 100 µM NADH (column 4).Controls without adducts assayed with or without the preincubationstep showed a slightly lower activity of InhA in the absenceof its cofactor (NADH), due to a partial denaturation.

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TABLE 3. Inhibition of InhA activity by an isolated pool of INH-NAD(H) adducts and by collected single INH-NAD(H) and INH-DNAD(H) adducts

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FIG. 5. Inhibition of InhA activity by an isolated pool of adducts collected from HPLC. Experiments were performed in triplicate except for concentrations 2 and 3 µM (one experiment). Error bars, standard deviations.

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FIG. 6. Protection of InhA activity by NADH or decenoyl-CoA against INH-NAD(H) adducts. Column 1, control experiment in the absence of adducts with 5-min preincubation of InhA (column 1; 100%); columns 2 to 4, remaining InhA activities when InhA was preincubated with either 100 nM of the isolated pool of adducts (column 2; 22%), 100 µM NADH (column 3; 104%), or 100 µM decenoyl-CoA (column 4; 90.5%). Experiments were performed in duplicate (errors of < 7%).

InhA assay. Enoyl reductase activity of InhA was assayed by monitoring theoxidation of NADH at 340 nm (25°C), and initial velocitieswere determined. Results are expressed as a percentage of remainingInhA activity determined on the basis of control experiments.

RESULTS AND DISCUSSION

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Results and Discussion

Mn(III) pyrophosphate-dependent oxidation of INH and formation of INH-NAD(H) adducts and related compounds. As shown in Table 1, the oxidation of INH by two equivalentsof Mn(III) pyrophosphate (run 1) gave a high conversion rateof INH (93%) within a few minutes [in the absence of Mn(III)pyrophosphate, conversion of INH was below 3%, even after 48h of incubation (run 2)]. The stoichiometry of this two-electronoxidation is consistent with that previously observed with Mn(III)malonate (13). This efficient chemical oxidation method gavethe same stable products, compounds 1 to 3 (Fig. 1), as thoseobtained with KatG (10). However, since none of these productsinhibits InhA (9), one key point remained the ability of theMn(III) pyrophosphate system to produce the short-life reactiveINH oxidation intermediates (e.g., the isonicotinoyl radicalor ion) involved in the formation of the adducts with the NAD⁺/NADHcofactor of InhA and to lead to the inhibition of this enzyme.

Thus, in a second step, we used Mn(III) pyrophosphate in thepresence of both INH and either NAD⁺, NADH, NMN⁺, or DNAD⁺ fortesting its ability to produce various INH-coenzyme adducts.Monitoring by HPLC, we observed in all cases (Table 1, runs3 to 6), in addition to the stable INH oxidation compounds 1to 3, the formation of a family of products with UV spectracharacteristic for dihydropyridine derivatives (absorbance peakat 320 to 333 nm), like those we recently observed with NAD⁺and DNAD⁺ (17) and also like those observed by Johnsson et al.with the use of the KatG/MnCl₂ as activating system (26). LC-ESI-MSanalyses showed that six products having the same molecularmass could be detected in reactions performed with either NADH(molecular weight, 770) or with NMN⁺ (molecular weight, 441),as it was also the case for the previously described adductswith NAD⁺ (molecular weight, 770). For DNAD⁺ adducts, only twoproducts with a mass of 771 were detected, as previously reported(17). The overall yield of the pool of adducts ranged from 35to 45% in the case of NAD⁺, depending on the reaction conditions,but was only 5 to 14% when NADH, NMN⁺, or DNAD⁺ was used. Basedon their HPLC behavior, UV data, and mass data, the six INHadducts formed either with NAD⁺ or NADH were shown to be identical.The most likely mechanism of INH-NAD(H) adduct formation consistsof the addition of an isonicotinoyl radical, produced by Mn(III)pyrophosphate INH oxidation, to the electron-deficient pyridiniumring of NAD⁺, following a Minisci reaction (16). The resultingradical cation then looses an electron to form the final adductproduct. The lower yield observed for the formation of adductsin the presence of NADH may be explained by an initial conversionof NADH into NAD⁺. Indeed, we observed that Mn(III) pyrophosphate(100 µM) alone was not able to oxidize NADH (100 µM),but after a 10-min incubation in the presence of INH (500 µM),NAD⁺ was formed in significant amounts (27% yield). Such NADHoxidation was also observed by using KatG/MnCl₂ as the oxidantsystem (26). The even-lower yields observed for INH adductswith NMN⁺ or DNAD⁺ (5 to 6% [Table 1]), while the conversionof INH was high (60 to 84%), evidenced the critical role ofstructural parameters such as the presence of the adenylic moietyor the amide function at position 3 of the pyridinium ring.

The multiple peaks observed in HPLC profiles might be ascribedto the coexistence in solution of cyclized diastereoisomers(cases of INH adducts with NADH and NMN⁺ are similar to thecase of adducts with NAD⁺) in addition to two epimeric openstructures (which were the only ones present for DNAD⁺ adducts)(17). Oxidized adducts with a pyridinium ring instead of thedihydropyridine structure were also present in small amounts(see peaks 10 and 13 in Fig. 2 and the corresponding structuresin Fig. 1; these products did not show an absorption peak around320 to 330 nm). Since compound 10, isolated in the case of NAD⁺,was shown to be devoid of any inhibitory activity (26), UV analysesat 320 to 333 nm (absorption wavelength of the dihydropyridinering) provide a rapid and convenient test for following theINH activation and detecting the INH-coenzyme adducts likelyto behave as inhibitors of InhA. In conclusion, the oxidationwith a stoichiometric amount of Mn(III) pyrophosphate appearsclearly to be an efficient and fast way of activating INH inorder to produce INH adducts in the presence of the nicotinicor nicotinamide coenzymes. These adducts represent potentialinhibitors of the InhA target.

Formation of adducts in the presence of InhA and simultaneous inhibition of the enzyme activity. We investigated the INH-dependent inhibition of InhA in thepresence of Mn(III) pyrophosphate and NADH, the natural cofactorof InhA (Fig. 4A; Table 2, runs 1 to 3) to verify that the adductsgenerated by this chemical activation of INH were efficientinhibitors. As shown in Fig. 4A and Table 2 (run 1), Mn(III)pyrophosphate efficiently mediated the inhibition of InhA activityby INH in the presence of NADH in a time-dependent process (79%loss of InhA activity after 12 min of incubation). Such a kineticprofile may correspond to either a slow kinetic of NAD⁺ formationfrom NADH prior to adduct formation (see discussion above) orto the time necessary to reach the equilibrium, since in theseexperiments, the adduct has to compete with an excess of NADHfor binding to the InhA active site. Significant inhibitionwas not observed when either Mn(III) pyrophosphate (run 2) orINH or both of these two reagents were omitted (Fig. 4A). Inthe absence of NADH (Fig. 4A; Table 2, run 3), partial time-dependentinhibition of InhA was observed (31% loss of InhA activity after12 min of incubation), possibly due to the instability of InhAin the absence of its cofactor. However, we could not rule outthe possibility of an InhA inactivation due to a nonspecificinteraction of activated INH species with the enzyme (e.g.,through alkylation of amino acid residues). In order to distinguishbetween these two possibilities, INH was first preincubatedwith Mn(III) pyrophosphate in the absence of InhA under thesame conditions described above. After preincubation at RT for18 min, the enzyme was added. No significant loss of activitywas observed in this way after a further 12-min incubation (datanot shown), arguing against any instability of the enzyme inthe absence of its cofactor but rather supporting a nonspecificinteraction of transient reactive INH oxidation intermediateswith the enzyme.

These results altogether show that it is possible to activateINH with a simple nonenzymatic process and, through the formationof INH adducts with the NADH cofactor of InhA, to nearly completelyinhibit this enzyme within a few minutes.

Inhibition of InhA by a pool of preformed adducts. We studied the inhibition of InhA by a pool of preformed adductsin two consecutive steps: first, we oxidized 100 µM INHin the presence of 100 µM NADH, NAD⁺, NMN⁺, or DNAD⁺ atRT under the same conditions described above for 18 min, a reactiontime sufficient for complete INH adduct formation; second, thereaction medium containing the preformed pool of INH adductswas mixed with 3 µM InhA to study the kinetic data. Theaim was to examine whether INH adducts produced in the absenceof InhA are able to show the same inhibition activity as thoseformed in the presence of InhA and to investigate the possiblerole of the enzyme target in formation of the inhibitors. Inthe curves corresponding to evaluation of INH adducts with NADHprepared in the presence (Fig. 4A; Table 2, run 1) and in theabsence of InhA (Fig. 4B; Table 2, run 4), we note a similarprofile, with a time-dependent inhibition. In the second experiment,the adducts were preformed, so no kinetic of adduct formationcan explain the time-dependent inhibition, which can be proposedin the first case. In addition, we have verified that after2 h of preincubation of INH, NADH, and Mn(III) pyrophosphate,the concentration of INH adducts was the same and that the subsequentinhibition kinetic profile of InhA was unchanged. Thus, thetime-dependent profile in Fig. 4B might correspond to the timenecessary to reach the equilibrium between binding of NADH andbinding of the competitive inhibitor to the active site. TheInhA inhibition appeared slightly lower with preformed adductsthan with adducts formed in the presence of InhA (Fig. 4; Table2, runs 4 and 1, respectively).

In the case of adducts obtained in the presence of the oxidizedcoenzyme NAD⁺, formed in higher yields (Table 1, run 3, 41%),the inhibition was very fast and almost complete (Fig. 4B; Table2, runs 7 to 9), even for short preincubation times (preincubationfor 2 min was sufficient to form adducts giving up to 86% inhibitionof InhA activity). Here, the initial higher concentrations ofthe inhibitory adducts and the known lower affinity of NAD⁺for InhA (compared to NADH) (21) probably allowed to quicklyreach the equilibrium.

INH adducts with other nicotinamide or nicotinic coenzymes weresimilarly evaluated as possible InhA inhibitors. NMN⁺ contrastswith NAD⁺ by the absence of the adenine nucleotide part andDNAD⁺ by the replacement of the carboxamide function of NAD⁺by a carboxylic group. In both cases (Fig. 4B; Table 2, runs11 and 13, respectively) and using strictly the same experimentalconditions as with NAD⁺, no InhA inhibition was observed. Thisabsence of reactivity, keeping however in mind the low levelof adducts yielded (Table 1), might indicate that both the nicotinamidefunction and the adenylic moiety play a crucial role in theinhibitor binding and contribute to its affinity for the InhAtarget.

These results mainly show that INH-NAD(H) adducts can be formedusing the Mn(III) pyrophosphate activation system in the absenceof InhA. These adducts have the same potency to inhibit InhAactivity as the adducts formed in the presence of the enzyme,signifying that preformed adducts are able to bind to the InhAactive site. The rather small differences of inhibition rateobserved between experiments in which adducts were either preformedor formed in the presence of InhA do not allow us to estimatewhether, in vivo, the inhibitory adduct forms itself betweenactivated INH and NAD(H) within the InhA active site or formsoutside the active site and then binds to it.

Inhibition of InhA by an isolated pool of adducts or by collected single adducts. The isomeric adducts described above are rather stable as longas they are kept as a mixture in the buffered reaction mediumat RT. One of the main difficulties in evaluating their biologicalactivity is related to separation and isolation problems. Asan example, when the main and well-separated compounds 6 and7 (Fig. 2A) were collected from HPLC and then lyophilized andredissolved in water, the subsequent reinjection of the collectedsamples in the HPLC column displayed a mixture of the differentinitial isomers as well as some unidentified degradation products.Such a phenomenon has already been observed (26). Salt and/orpH effects during concentration of samples might explain thisbehavior, with a possible epimerization occurring at position4 through a pH-dependent keto-enolic equilibrium. Hence, weestablished a protocol allowing us to test the inhibition ofInhA activity directly from the fractions collected from HPLC.Three kinds of experiments were performed: (i) one with a poolof INH-NAD(H) adducts isolated by HPLC, (ii) one with the isolatedcompounds 6 and 7, and (iii) one with the isolated compounds11 and 12. Adducts formed in amounts that were too small werenot collected. Adduct concentrations of the collected HPLC fractionswere determined using a ${varepsilon}$ ₂₆₀ of 27 mM^-1 cm^-1, a value estimatedby Lei et al. for an INH-NAD(H) adduct (11). The same ${varepsilon}$ valuewas used for INH-DNAD(H) adducts 11 and 12, considering thatthe absorption of these compounds should be close to that ofINH-NAD(H) adducts (the starting cofactors NAD⁺ and DNAD⁺ havethe same ${varepsilon}$ ₂₆₀, 18 mM^-1 cm^-1).

The inhibition of InhA by increasing concentrations of the poolof adducts is displayed in Fig. 5 and shows a dose-dependentinhibition. From the crystallographic data (22) on the structureof the complex InhA/INH-NAD(H) adduct, the NAD part of the adductlogically binds to the NADH binding site and the isonicotinoylmoiety binds to the substrate binding site. Thus, the completeadduct must compete with NADH and possibly with the enoyl substratesfor binding to the InhA active site. This point is supportedby results shown in Fig. 6. An isolated 100 nM pool of INH-NAD(H)adducts first incubated for 5 min with 77 nM InhA was shownto give a 78% inhibition of the enzyme activity (column 2).This inhibition was completely (column 3) or almost completely(column 4) reversed when the inhibitor was only added afterpreincubation of InhA in the presence of 100 µM NADH or100 µM decenoyl-CoA, respectively. This phenomenon ofprotection by the NADH cofactor or the enoyl-CoA substrate explainsalso why, under the conditions of Fig. 5, where the inhibitorhas to compete with both compounds, the concentration of INH-NAD(H)adducts necessary to observe an inhibition of about 80% wasclearly higher (about 5 µM).

Concerning the inhibition observed with the isolated adductforms, results shown in Table 3 indicate that compound 6 wasclearly more efficient than compound 7 and was as efficientas the total pool. Among DNAD⁺ adducts, adduct 12 was a poorinhibitor, while adduct 11 was devoid of any inhibitory activity.Considering the adduct formula shown in Fig. 7, compounds 11and 12 have the two possible configurations R and S at position4 and were formed in a ratio of nearly 1:1. So, the alkylationprocess of the coenzyme by the activated form of INH must beconsidered to occur equally on each face of the pyridinium ring.Compounds 6 and 7, and similarly the pair of compounds 8 and9, were also formed in similar amounts, and we can suppose thateach pair contains one R isomer and one S isomer at the C-4position. Since the pair of compounds 6 and 7 was formed inmuch higher yield than the pair of compounds 8 and 9, one hypothesisis that the cyclization process occurs preferentially with thetwo bulky substituents at C-4 and C-7 in trans position (Fig.7). So, the compound pair 6-7 might be the couple of isomers4S,7R and 4R,7S, and the compound pair 8-9 might be the couple4S,7S and 4R,7R. A more precise attribution for compounds 6and 7, as also for compounds 11 and 12, might be deduced fromthe biological results (Table 3). The most-likely structurefor the most-active compounds 6 and 12 should be that with configurationS at C-4, which is the configuration proposed for the boundinhibitor present in the crystal structure of the InhA-inhibitorcomplex (22). Taken together, these arguments led us to proposefor each of the compounds 6, 7, 11, and 12 the absolute configurationsat C-4 and C-7 as ascribed in Fig. 7.

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FIG. 7. Structures of compounds 6 to 9 and compounds 11 and 12 and proposed stereochemistry of chiral centers for compounds 6, 7, 11, and 12. Adapted from reference 17.

It is noteworthy that one of the cyclic forms of the INH-NAD(H)adduct (form 6) has a relatively strong inhibitory potency onInhA activity, while the crystal structure of the adduct boundto InhA in the binary complex InhA-adduct was interpreted asan open form (Fig. 3); (22). Thus, it is legitimate to raisethe question about the effective active form of the INH-NAD(H)adduct(s). Is there a single active form, or are several formsable to inhibit the InhA activity, with different levels ofinhibitory potency?

In conclusion, we provide a method for a very fast and efficientinhibition of InhA in the presence of NAD⁺/NADH and INH, usingMn(III) pyrophosphate as a simple nonenzymatic oxidative INHactivator. This chemical oxidant provides a simple way to synthesizevarious INH derivatives, mimicking the bioactivation pathwayof this drug in M. tuberculosis. The structure-activity relationshipsof the different types of INH-coenzyme adducts evidenced inthis work will be useful to facilitate the design of new inhibitorsof InhA, with potential use as antituberculous drugs.

ACKNOWLEDGMENTS

We thank Catherine Claparols and Suzy Richelme for their collaborationon all ESI-MS analyses, which were performed at the UniversityPaul Sabatier in the Service de Spectrométrie de MasseFR14-LCC. We thank Gilbert Laneelle and Mamadou Dafféfor helpful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse Cedex 4, France. Phone: 33 (0)5 61 33 31 46. Fax: 33 (0)5 61 55 30 03. E-mail: bernadou{at}lcc-toulouse.fr.

REFERENCES

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