Brief Article pubs.acs.org/jmc
Discovery of Covalent Inhibitors of Glyceraldehyde-3-phosphate Dehydrogenase, A Target for the Treatment of Malaria Stefano Bruno,*,†,∥ Andrea Pinto,‡ Gianluca Paredi,† Lucia Tamborini,‡ Carlo De Micheli,‡ Valeria La Pietra,§ Luciana Marinelli,§ Ettore Novellino,§ Paola Conti,*,‡ and Andrea Mozzarelli†,⊥ †
Dipartimento di Farmacia and Centro Siteia.Parma, Università di Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy § Dipartimento di Farmacia, Università di Napoli Federico II, Via Montesano, 49, 80138 Napoli, Italy ∥ Istituto di Bioscienze e Biorisorse, CNR, 80131 Napoli, Italy ⊥ Istituto Nazionale di Biostrutture e Biosistemi, 00136 Roma, Italy ‡
S Supporting Information *
ABSTRACT: We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis.
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INTRODUCTION Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) is a key glycolytic homotetrameric enzyme that catalyzes the conversion of glyceraldehyde 3-phosphate (G3P) into 1,3-biphosphoglycerate (1,3-BPG), with the concomitant reduction of nicotinamide adenine dinucleotide (NAD+) to NADH.1 The reaction mechanism consists of two steps: (i) an oxidoreduction, with the nucleophilic attack of the catalytic Cys on the aldehydic group of G3P, followed by a hydride transfer from the thiohemiacetal to the C4 of the nicotinamide ring and (ii) the phosphorolysis of the resulting thioester through the nucleophilic attack of inorganic phosphate. The second step is preceded by the exchange of NADH with NAD+, which favors phosphorolysis. The structure of the enzyme from different sources has been determined and its reactivity in the crystalline state characterized.2−4 GAPDH is a potential antiparasitic target because, in the amastigote-phase, several pathogenic protozoans rely solely on glycolysis for energy production. Inhibitors of the enzymes from Trypanozoma cruzi,5,6 Leishmania mexicana,7 and Trypanosoma brucei8,9 have been identified, in some cases exhibiting selectivity toward the parasitic enzyme with respect to the human homologue.10 We specifically focused on GAPDH from Plasmodium falciparum (Pf GAPDH), which possesses a characteristic large channel connecting the bulk phase to the catalytic Cys (Supporting Information (SI) Figure S1).11,12 As it was recently re-emphasized,13 the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. GAPDH is inactivated by a number of compounds that alkylate thiols.14,15 3-Bromo-isoxazolines © XXXX American Chemical Society
exhibited this reactivity toward cytidine triphosphate synthetase (CTPS), a glutamine amido transferase endowed with a catalytic Cys within the active site.16,17 It was demonstrated that bromine is a better leaving group than chlorine, 3-Bracivicin 1 (Figure 1) being about 3-fold more potent than its chloro-analogue, the natural antibiotic acivicin 2.17 We herein evaluated the strength of this warhead for the irreversible inactivation of Pf GAPDH by testing 1, 2, and a series of
Figure 1. Model and target derivatives. Received: May 14, 2014
A
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potential inhibitors 3−12, with distinct substitution patterns at the 5 position to modulate reactivity (Figure 1).
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RESULTS AND DISCUSSION Chemistry. The synthesis of all the derivatives was based on the 1,3-dipolar cycloaddition of bromonitrile oxide, generated in situ by dehydrohalogenation of the stable precursor dibromoformaldoxime (DBF)18 to the suitable dipolarofile, affording in all cases the sole 5-substituted 3-bromo-isoxazoline derivative. Derivatives (S,S)-1,17 (S,S)-2,19 (±)-7,16 (±)-8,20 (±)-9,21 (+)-9,22 (−)-9,22 and (S,S)-1016 were previously described, whereas derivatives (±)-3−6 and (S,S)-11−12 were obtained according to Scheme 1. Cycloaddition of bromoni-
Figure 2. Representative time courses of Pf GAPDH inactivation at 33 nM by (S,S)-10 (A) and (±)-8 (B) at different inhibitor concentrations. For (S,S)-10, concentrations of 200 (squares), 100 (diamonds), 70 (triangles), and 50 μM (circles) were used. For (±)-8, concentrations of 300 (squares), 100 (diamonds), 70 (triangles), and 50 μM (circles) were used. The activity was normalized to that in the absence of inhibitors. The lines are the fittings to a sum of two exponential decays. All experiments were carried out in duplicate.
Scheme 1. Synthesis of 3-Bromo-isoxazoline Derivativesa
The full inhibition curves exhibited a well-defined biphasicity (Figure 2), with the relative amplitude of the two phases depending on the specific compound and its concentration (SI Figure S2). The first fast phase was completed within a few minutes, whereas the second was completed in time frames ranging from several minutes to several hours, depending on the inhibitor concentration. Because enzyme inhibition is only due to alkylation of Cys153 (see below), the biphasicity suggests the existence of at least two distinct conformations of the enzyme subunits, with an approximately 20-fold difference in reactivity, within an apparently structurally symmetric tetramer.23 Half-of-sites reactivity of GAPDH has been evidenced by exploiting the chromophoric substrate analogue β-(2-furyl)-acryloyl-phosphate (FAP)24,25 but not with natural substrates.26 The dependence of the amplitude of the two phases on inhibitor concentration suggests that its binding affects conformational equilibria (SI Figure S3). We ruled out the possibility that biphasicity arises from enantiomers in racemic mixtures by monitoring the inactivation by pairs of enantiomers (SI Figure S4). The dependence of the rate constants of both the fast (SI Figure S5) and the better characterized slow phase (Figure 3) on the concentration of inhibitor was linear in a Kitz−Wilson double-reciprocal plot, as expected for a bimolecular covalent inhibition.27 The reaction parameters were determined by fitting the dependencies to eq 1, which describes affinity labeling.
a
Reagents and conditions: (a) DBF, NaHCO3, EtOAc, rt, 12 h; (b) 10 N HCl/THF, rt, 4 h; (c) TMSCHN2, toluene/MeOH, rt, 1 h; (d) BnBr, NaI, K2CO3, DMF, 50 °C, 1 h; (e) 30% TFA/CH2Cl2, 0 °C → rt, 4 h.
trileoxide to alkenes 13−15 afforded the 3-bromo-isoxazoline derivatives (±)-3, (±)-5, and (±)-6, respectively. Chloroderivative (±)-4 was obtained from the corresponding bromoderivative (±)-3 by treatment with 10 N HCl in THF, following the procedure previously described for the synthesis of (S,S)-2.19 Derivative (S,S)-11 was obtained by direct esterification of carboxylic acid (S,S)-1616 with trimethylsilyldiazomethane, followed by Boc deprotection with 30% TFA in dichloromethane. In parallel, (S,S)-12 was obtained from (S,S)16 by treatment with benzyl bromide in the presence of K2CO3 and a catalytic amount of NaI, followed by Boc deprotection. Inhibition Kinetics. Compounds 1−12 were assayed as Pf GAPDH inhibitors. The 3-bromo-isoxazoline derivatives brought about a time- and concentration-dependent inactivation of Pf GAPDH, with a large variability in reaction rates (Figure 2). Conversely, the 3-chloro-isoxazoline derivatives (±)-2 and (±)-4 produced slow inactivation, with completion times of at least 1 order of magnitude higher, preventing their full characterization. The comparison of 3-bromo and 3-chloroisoxazoline rings exhibiting the same substituents (i.e., (S,S)-1 vs (S,S)-2 and (±)-3 vs (±)-4) led to the conclusion that the latter are intrinsically far less reactive, as previously observed for CTPS.17 Extensive dialysis of the enzyme solutions after inactivation did not restore activity, confirming an irreversible inhibition mechanism.
kobs =
k inact[I] K i + [I ]
(1)
Specifically, the y-intercept is kinact, the maximum rate of inactivation for each compound at saturating concentration, whereas the slope kinact/Ki is the apparent second-rate constant of inactivation. The close-to-zero intercepts calculated for all inhibitors (Figure 3 and SI Figure S5) indicated that the initial noncovalent binding do not contribute to inhibition. The inhibitors are listed in Table 1, ranked on the basis of the kinact/ Ki ratio, higher values indicating higher inhibitory potency.27 Assuming a negligible contribution by the noncovalent binding phase, a 20-fold difference in kinact/Ki indicated that the most active compound, (S,S)-11, with a kinact/Ki of 10.7 ± 2.8 M−1 s−1, inhibits the enzyme 20 times faster than the least B
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MALDI-TOF analysis of undigested Pf GAPDH (Figure 4A) resulted in a molecular mass of 39950 ± 10 Da, as assessed
Figure 3. Kitz−Wilson double reciprocal plot of the inhibition of Pf GAPDH by selected 3-bromo-isozaxoline compounds: (±)-5 (open diamonds), (S,S)-1 (open squares), (±)-7 (closed diamonds), (±)-8 (open upward triangles), (S,S)-10 (crosses), (±)-6 (downward closed triangles), (±)-9 (upward closed triangles), (±)-3 (open circles), (S,S)-11 (closed circles) (S,S)-12 (closed squares). All experiments were carried out at least in duplicate.
Figure 4. (A) Mass spectra of the double charged peak of undigested Pf GAPDH (black line) and upon overnight incubation with (S,S)-11 at 200 μM concentration (gray line). (B−C) MS of the tryptic digests of the unmodified protein (B), with peptide A (*) at 1947 m/z ([M + H]+) and (C) of the tryptic digest of the protein upon incubation with (S,S)-10, with peptide A (*) at 2280 m/z ([M + H]+), corresponding to alkylation with both iodoacetamide and (S,S)-10.
Table 1. Kitz−Wilson Parameter for Selected 3-Bromoisozaxoline Compounds compd (S,S)-11 (±)-3 (S,S)-12 (±)-9 (±)-6 (±)-8 (S,S)-10 (±)-7 (S,S)-1 (±)-5
from the doubly charged ion, consistent with the loss of the initial methionine (expected molecular monoisotopic mass: 39959 Da). A secondary peak (72 °C dec; Rf = 0.34 (EtOAc). 1H NMR (300 MHz, CDCl3) δ 3.12 (dd, J = 8.0, 17.3 Hz, 1H), 3.70 (dd, J = 11.0, 17.3 Hz, 1H), 5.65 (dd, J = 8.0, 11.0 Hz, 1H), 7.22 (d, J = 6.0 Hz, 2H), 8.60 (d, J = 6.0 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 49.2, 81.3, 120.6, 137.0, 148.7, 150.5.MS: 227.1 [M + H]+. Anal. (C8H7BrN2O) C, H, N. 4-(3-Bromo-4,5-dihydroisoxazol-5-yl)phenol [(±)-6]. Yield 78% (yellow oil); Rf = 0.44 (cyclohexane/EtOAc, 7:3). 1H NMR (300 MHz, CDCl3) δ 3.20 (dd, J = 9.6, 17.3 Hz, 1H), 3.55 (dd, J = 9.6, 17.3 Hz, 1H), 5.60 (dd, J = 9.6, 9.6 Hz, 1H), 6.50 (bs, 1H), 6.85 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 49.0, 83.7, 116.1, 128.1, 130.7, 137.5, 156.8. MS: 241.8 [M + H]+. Anal. (C9H8BrNO2) C, H, N. Synthesis of 3-Chloro-5-phenyl-4,5-dihydroisoxazole [(±)-4]. (±)-3 (2.0 mmol) was dissolved in a 10 N THF solution of HCl (5 mL). The reaction mixture was stirred for 4 h. The solvent was evaporated, water (5 mL) added, and the aqueous phase extracted with EtOAc (2 × 5 mL). The organic extracts were dried over anhydrous Na 2 SO4 and evaporated under reduced pressure. Purification of the crude material by column chromatography afforded the desired Cl-derivative (±)-4. Yield 90% (colorless oil); Rf = 0.46 (cyclohexane/EtOAc, 9:1). 1H NMR (300 MHz, CDCl3) δ 3.18 (dd, J = 9.1, 17.0 Hz, 1H), 3.62 (dd, J = 10.7, 17.0 Hz, 1H), 5.67 (dd, J = 9.1, 10.7 Hz, 1H), 7.35−7.42 (m, 5H). 13C NMR (75 MHz, CDCl3) δ 46.4, 84.1, 126.2, 129.0, 129.2, 139.5, 148.8. MS: 182.0 [M + H]+. Anal. (C9H8ClNO) C, H, N. Synthesis of (S)-Methyl 2-((S)-3-Bromo-4,5-dihydroisoxazol5-yl)-2-(tert-butoxycarbonylamino)acetate [(S,S)-17]. At 0 °C, E
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Boltzmann aqueous solvation model35 and otherwise default settings were selected.
enzyme activity determined. An aliquot of Pf GAPDH was maintained as a control under the same conditions but in the absence of inhibitors. Typically, less than 5% of activity loss in the control sample was observed within 6 h. The fractional inactivation kinetics were analyzed either as a mono- or biexponential decay. The dependence of the resulting rate constants of the better resolved slow phase at different inhibitor concentrations was used to rank the inhibitors. On the basis of the working hypothesis of a mechanism-based inhibition, with negligible hydrolysis of the product, the following inhibition scheme was applied:
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ASSOCIATED CONTENT
S Supporting Information *
Details of the methods used and additional analysis of mass spectrometry data. This material is available free of charge via the Internet at http://pubs.acs.org.
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E + IX → EIX → E−I + X
AUTHOR INFORMATION
Corresponding Authors
with E the enzyme, I the inhibitor, X the leaving group, EIX the noncovalent complex between E and IX, and E−I the covalent, inactive complex. The apparent inhibition constants kobs were determined by fitting the dependence of the residual activity versus time of incubation with eq 1, describing a mechanism-based inhibition. kinact is the rate of enzyme inactivation, and Ki is the dissociation constant of the initial noncovalent EI complex. Absorption Spectroscopy. Absorption spectra of GAPDH28 were measured using a Varian CARY400 spectrophotometer. To evaluate the disappearance of the Racker band, the protein at 400 μM concentration was incubated with (±)-3 and absorption spectra were collected until completion of the reaction. Mass Spectrometry. Mass spectrometry was carried out using a 4800 Plus MALDI TOF/TOF AbSciex and a LTQ-Orbitrap. Undigested proteins were loaded onto the MALDI plate with the double layer method. Briefly, 1 μL of 10 mg/mL α-cyano-4hydroxycinnamic acid (CHCA) in acetone was deposited onto the MALDI plate and air-dried. A second layer was obtained by mixing an equal volume of sample with CHCA in acetonitrile 50%−TFA 2.5%. The digested proteins were analyzed with thin layer methods using CHCA in acetonitrile 50%−TFA 0.05%. MS/MS spectra were collected with the same protocol. The reactivity toward iodoacetamide was assessed by incubating Pf GAPDH at 20 μM concentration with a 10-fold excess of iodoacetamide for 1 h, followed by MALDI TOF analysis of either the tryptic digest or the undigested protein. A similar approach was used for the evaluation of the binding of the 3-bromoisoxazoline compounds, varying the time of incubation depending on the experimentally determined inhibition rates. To prevent nonspecific alkylation to Cys residues which are not solvent-exposed once the protein has been denatured, the reaction mixture was dialyzed three times to remove the excess of alkylating agents. Tryptic digestion was carried out using DigestPro MSi intavis. Analysis of mass spectra was carried out using mMass 5.5. Computational Chemistry. The 3D structures of the compounds were generated with the Maestro Build Panel and prepared with the Ligprep module which automatically generates the protonation state at pH 7 ± 1.31 The target Pf GAPDH structure (PDB 2B4R) was prepared through the Protein Preparation Wizard of the graphical user interface Maestro 9.2.11231 and the OPLS-2001 force field. Water molecules were removed, and hydrogen atoms were added, while the cofactor NAD+ has been retained into the protein. Covalent docking was carried out using the Schrodinger protein modeling and refinement package Prime.32 Here OPLS2005 force field with implicit solvation and a distance-dependent dielectric of 2R was employed. The exploration of the conformational space of the ligand and Cys153 was done using a procedure similar to that employed in loop refinement in Prime.33 Molecular Electrostatic Potential Calculations. For all compounds, the electrostatic potential was calculated by means of Jaguar 7.834 and mapped onto the electron density surface for each compound. The isovalue of 0.01 electron/Bhor3 was chosen for the definition of the density surface, while the electrostatic potential was computed using HF functional and LACVP* and 6-31G* basis sets with a scale of −79 (red) to 100 hartree (purple), −10.5 and 10.5 kcal mol−1, respectively. The LACVP* basis set uses the standard 6-31G* basis set for light elements and the LAC pseudopotential (effective core potential for inner electrons) for the Br atom. The Poisson−
*For S.B.: phone, (+39)0521906613; E-mail, stefano.bruno@ unipr.it. *For P.C.: phone, (+39)0250319329; E-mail, paola.conti@ unimi.it. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from MIUR (PRIN 2012) and from Ministero degli Affari Esteri are gratefully acknowledged. ABBREVIATIONS USED CHCA, α-cyano-4-hydroxycinnamic acid; CTPS, cytidine triphosphate synthetase; DBF, dibromoformaldoxime; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); FAP, β-(2-furyl)-acryloylphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase
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