proteins STRUCTURE O FUNCTION O BIOINFORMATICS

Tamoxifen, an anticancer drug, is an activator of human aldehyde dehydrogenase 1A1 Javier A. Belmont-Dıaz, Luis F. Calleja-Casta~ neda, Belem Yoval-Sanchez, and Jose S. Rodrıguez-Zavala* Departamento de Bioquımica, Instituto Nacional de Cardiologıa, Mexico D.F, Mexico

ABSTRACT The modulation of aldehyde dehydrogenase (ALDH) activity has been suggested as a promising option for the prevention or treatment of many diseases. To date, only few activating compounds of ALDHs have been described. In this regard, N-(1,3-benzodioxol-5-ylmethyl)22,6-dichlorobenzamide has been used to protect the heart against ischemia/reperfusion damage. In the search for new modulating ALDH molecules, the binding capability of different compounds to the active site of human aldehyde dehydrogenase class 1A1 (ALDH1A1) was analyzed by molecular docking, and their ability to modulate the activity of the enzyme was tested. Surprisingly, tamoxifen, an estrogen receptor antagonist used for breast cancer treatment, increased the activity and decreased the Km for NAD1 by about twofold in ALDH1A1. No drug effect on human ALDH2 or ALDH3A1 was attained, showing that tamoxifen was specific for ALDH1A1. Protection against thermal denaturation and competition with daidzin suggested that tamoxifen binds to the aldehyde site of ALDH1A1, resembling the interaction of N-(1,3-benzodioxol-5ylmethyl)22,6-dichlorobenzamide with ALDH2. Further kinetic analysis indicated that tamoxifen activation may be related to an increase in the Kd for NADH, favoring a more rapid release of the coenzyme, which is the rate-limiting step of the reaction for this isozyme. Therefore, tamoxifen might improve the antioxidant response, which is compromised in some diseases. Proteins 2015; 83:105–116. C 2014 Wiley Periodicals, Inc. V

Key words: aldehyde dehydrogenases; ALDH1A1; tamoxifen; oxidative stress; lipid peroxidation products.

INTRODUCTION Oxidative stress has been related to the etiology and pathogenesis of different diseases of increasing incidence caused by poor eating habits and sedentary lifestyle. These diseases include: (i) Cardiovascular problems culminating in ischemic events where the blood flow to the organ (commonly heart or brain) is interrupted, followed by a re-establishment of the blood flow (reperfusion), which generates a burst of reactive oxygen species damaging the cells1–3; (ii) Other diseases related to metabolic syndrome such as obesity, diabetes, atherosclerosis, hypertriglyceridemia, fatty liver disease, among others4–6; (iii) Neurodegenerative disorders such as Alzheimer, Parkinson, multiple sclerosis, among others7–11; (iv) Intoxication with heavy metals originating an increase in reactive oxygen species due to redox imbalance12,13; and (v) Oxidative stress is also involved in the pathogenesis of different ocular diseases14–16 and cancer.17–19 During severe events of oxidative stress, the primary antioxidant defense barriers (glutathione, catalases,

C 2014 WILEY PERIODICALS, INC. V

superoxide dismutases, peroxidases, and glutathione Stransferases) may be overwhelmed leading to membrane lipid peroxidation. This process gives rise to a wide variety of aldehydes, and some of the most important due to their toxicity are malondialdehyde, 4-hydroxy-2-nonenal, 4-hydroxy-2-hexenal, and acrolein.20–22 The toxic effect of aldehydes is related to their high reactivity forming adducts with enzymes causing inactivation and with DNA that may result in mutagenesis.21,22 Additional Supporting Information may be found in the online version of this article. Abbreviations: ALDA-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide, activator of human ALDH2; ALDH1A1, human aldehyde dehydrogenase class 1A1; ALDH2, human aldehyde dehydrogenase class 2; ALDH3A1, human aldehyde dehydrogenase class 3A1; DOPAL, 3,4-dihydroxyphenylacetaldehyde. Grant sponsor: CONACyT Mexico; Grant number: 166463. *Correspondence to: Jose Salud Rodrıguez-Zavala, Departamento de Bioquımica, Instituto Nacional de Cardiologıa, Juan Badiano No. 1, Seccion XVI, Tlalpan, Mexico D.F. 14080, Mexico. E-mail: [email protected] or [email protected] Received 25 June 2014; Revised 5 October 2014; Accepted 18 October 2014 Published online 30 October 2014 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24709

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Aldehyde dehydrogenases (ALDHs) are a superfamily of enzymes that catalyze the irreversible oxidation of aldehydes to their corresponding acids using NAD(P)1 as the coenzyme. These enzymes have different quaternary structures; some are dimers and others are tetramers. ALDHs have many differences in their primary structures, but the structure of the monomer is highly conserved among the members of the family.23–25 The monomer is formed by three domains: the coenzyme binding domain, the catalytic domain, and the oligomerization domain.23–25 The coenzyme and aldehyde binding sites have separate access paths; while the coenzyme binding domain is located at the surface of the monomer, the aldehyde enters the catalytic domain through a hydrophobic funnel, located opposite to the NAD1 binding site, near the interface of the two dimers in the case of the tetramer [Fig. 1(A)].24,25 Residues that form the funnel are believed to be responsible for the substrate specificity of each enzyme. The coenzyme binding domain communicates with the catalytic domain by a tunnel, where the transfer of hydride from the Cys302thiohemiacetal intermediate to the pyridine ring of NAD1 takes place as part of the reaction mechanism.26 ALDHs have an important role protecting the cells against the toxic effects of aldehydes.27 ALDH1A1 and human aldehyde dehydrogenase class 3A1 (ALDH3A1) have been related to the protection of the eye against lipid peroxidation products generated by the constant exposure to light.28–31 In addition, ALDH1A1 has a preponderant role in the synthesis of retinoic acid, indispensable for cell differentiation and development.32,33 On the other hand, ALDH1A1 has been proposed as a general marker for normal and malignant stem cells,34 and this enzyme may play important functional roles related to self-protection, differentiation, and expansion in these cells.35 Human aldehyde dehydrogenase class 2 (ALDH2) is involved in the protection of the heart against ischemia/reperfusion damage36–38; the activation of this enzyme has been proposed as a suitable therapeutic strategy to protect the heart during ischemic events.37–39 Additionally, the low activity of the ALDH2*2 variant is responsible for the ethanol flushing reaction due to acetaldehyde toxicity in East Asian populations, and the presence of this variant increases risk for late onset Alzheimer disease40,41 and development of different types of cancer.42–45 The progression of a number of neurological disorders such as Parkinson disease has also been correlated with the impaired activity of different enzymes of the ALDH superfamily.46–48 Similarly, a deficiency in succinate semialdehyde dehydrogenase (ALDH5A1) has been associated with epilepsy and intellectual disability.49,50 On the other hand, a role of ALDH2 in the bioactivation of nitroglycerin was recently elucidated,51 and the development of resistance against this compound, caused by prolonged treatment, was attributed to the

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irreversible ALDH2 inactivation induced by the nitroglycerin metabolism.51–53 Thus, the design of modulators of the ALDHs activity may have relevant biomedical applications. To the present day, only few activators of ALDHs have been reported, such as ALDA-1 (N-(1,3-benzodioxol5-ylmethyl)22,6-dichlorobenzamide), which activates ALDH1A154 and ALDH2,39 2-(benzo[d][1,3]dioxol-5yl)-N-(5,6-dihydro-4H-cyclopenta[c]isoxazol-3-yl) acetamide activator of ALDH1A1 found by virtual screening,54 and ALDA-89 (5-allyl-1,3-benzodioxole) specific for ALDH3A1.55 ALDA-1 has been used with success to prevent the damage to the heart during ischemia/reperfusion episodes38,39,56 and protects ALDH2 against inactivation by repeated administrations of nitroglycerin.57 The activator 2-(benzo[d][1,3]dioxol-5-yl)-N-(5,6-dihydro-4H-cyclopenta[c]isoxazol-3-yl) acetamide was found to activate ALDH1A1 and to protect the enzyme against inactivation by DOPAL (3,4-dihydroxyphenylacetaldehyde),54 an endogenous biogenic toxic aldehyde produced in catecholaminergic neurons,58 and ALDA-89 protects the submandibular gland function after radiotherapy.59 In the present work, the description and characterization of a new activating compound specific for human ALDH1A1 is reported.

MATERIALS AND METHODS Chemicals

All compounds were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Enzymes Nde I, Hind III, T4-ligase, and Vent DNA polymerase were from New England Biolabs (Ipswich, MA). Chelating Sepharose fast flow was from GE Healthcare (Piscataway, NJ). Expression and purification of the recombinant enzymes

The plasmid pT7-7 containing the DNA encoding the human ALDH1A1 or ALDH3A1 proteins flanked by an N-terminus His-Tag were used for the over-expression of the proteins. Full-length human ALDH2 cDNA was purchased from American Type Culture Collection (Manassas, VA; Cat. No. MGC-1806, GenBank ID: BC002967) and subcloned in the over-expression plasmid pT7-7 as previously described.60 E. coli BL21 (DE3)pLysS strain was transformed with the vectors containing the His-Tag constructs. The proteins were over-expressed and purified as reported previously.61,62 Briefly, the cells were harvested by centrifugation at 5000g, washed twice with 100 mL saline solution, re-suspended in a buffer containing 50 mM H2NaPO4 (pH 7.5), 500 mM NaCl, and 0.01% 2-mercaptoethanol, and then disrupted by sonication at 4 C. The extract was centrifuged at 100,000g for 30 min, and the supernatant containing the recombinant

Figure 1 Binding of ALDA-1 to ALDH2 (A and B) and tamoxifen to ALDH1A1 (C). (A) Structure of the tetramer of ALDH2 with ALDA-1 and NAD1 bound in the active site. ALDA-1 and NAD1 binding sites are highlighted by shadows in subunits A (colored green) and C (colored magenta), and the entrance to the respective binding sites is indicated with arrows. A zoom of the active site of subunit A in shown in a box to magnify the details. (B and C) Section (a): upper view of ALDA-1 (B) and tamoxifen (C) bound to ALDH2 and ALDH1A1, respectively, represented as surface; color codes: C, green; H, light gray; N, blue; O, red; S, yellow. Section (b): lateral view of a slice of subunit A of ALDH2 (B) and ALDH1A1 (C) represented as surface, showing the coupling of ALDA-1 and tamoxifen, respectively, in the entrance of the aldehyde binding site; part of the nicotinamide moiety of NAD1 represented as sticks is also shown. Color codes: C, gray; H, light grey, N, blue; O, red; S, yellow. Section (c): interaction of ALDA-1 (B) and tamoxifen (C) with residues of the aldehyde binding site shown as sticks; Cys302 and NAD1 are also shown in the figure. Color codes: C, grey; H, light gray; N, blue; O, red; S, yellow; P, orange. ALDA-1 and tamoxifen are colored cyan, and propionaldehyde in panel (C) is shown in green.

J.A. Belmont-Dıaz et al.

protein was applied to a 10-mL Chelating Sepharose column packed with NiCl2, equilibrated with the buffer described above at 4 C. The column was washed with 50 mM imidazole in the same buffer, and the protein was eluted applying a 100-mL total volume of a 50– 500 mM linear imidazole gradient. Fractions with activity were pooled, and ALDH1A1 and ALDH2 were concentrated using Amicon filters of 50,000 kDa MWCO, whereas ALDH3A1 was concentrated using Amicon filters of 30,000 kDa MWCO. Then, the enzymes were washed with buffer composed of 100 mM H2NaPO4 (pH 7.5), 100 mM NaCl, and 0.025% 2-mercaptoethanol to eliminate imidazole. Pure enzymes were concentrated and used fresh or stored in the presence of 50% glycerol at 220 C; before use, glycerol was washed out from the preparation by filtering the protein with assay buffer. Proteins were stable for more than 6 months in the presence of glycerol. The protein concentration was determined with the bicinchoninic acid protein assay kit (Sigma-Aldrich), using bovine serum albumin as standard. The enzyme purities were higher than 95% as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.63 Docking analysis

Data of the structures of ALDH1A1, ALDH2, and ALDH3A1 were obtained from the protein data bank (accession numbers 1BXS, 1CW3, and 1AD3, respectively). The generation and optimization of the threedimensional models of the compounds used in the study was conducted using the softwares ArgusLab 4.0.1 (available at: http://www.arguslab.com)64 and Maestro, version 9.1 (Schr€ odinger, LLC, New York, NY, 2010). Docking analysis was performed removing ligands from ALDH structures by using the software UCSF Chimera package 1.5.2 (Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, CA; supported by NIH P41 RR001081).65 ALDH structures and the ligand models were prepared for docking, using the software ADT 1.5.2.66 Docking analysis of ALDHs with tamoxifen was carried out using the software Autodock 4.2.5.1 (available at: http://autodock. scripps.edu/).67 One hundred conformations for each compound were obtained after docking, and then clustered for the analysis using ADT software. The conformations selected yielded the lowest values of binding energy and Ki and are presented in Table I and Supporting Information Table S1. The molecule of propionaldehyde was docked in the ALDH1A1 structure containing NAD1 and tamoxifen coupled to the active site, as described above, to generate sections (b) and (c) of panel (C) of Figure 1. Analysis of the resulting structures and generation of figures was achieved with PyMOL (The PyMOL Molecular Graphics System, Version 1.2.r1, Schr€ odinger, LLC).

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Table I Predicted Binding Energy and Kd Values of ALDH1A1, ALDH2, and ALDH3A1 with Tamoxifen Obtained by Docking Analysis ALDH1A1

ALDH2

ALDH3A1

9 26.9

657 24.4

4300 23.23

Kd (mM) Binding energy (Kcal/mol)

Activity assays

About 15–30 lg of protein was added to a buffer containing 100 mM H2NaPO4 (pH 7.4), 100 mM NaCl, 0.025% 2-mercaptoethanol, and 200 mM NAD1. The reaction was initiated by addition of 200 mM aldehyde. The ALDH activity was measured using a Shimadzu UV1800 spectrophotometer, following the increase in absorbance at 340 nm due to the formation of NADH. Stock solutions of aldehydes were routinely calibrated by enzymatic assay in the presence of a saturating concentration of NAD1. Daidzin inhibition assays were carried out in the above described buffer in the absence or the presence of 15 mM tamoxifen. NADH was used as inhibitor against NAD1 and propionaldehyde, to evaluate the inhibition patterns in the absence and presence of tamoxifen, to investigate a possible change in the kinetic mechanism of the enzyme.68 The Kiq for ALDH1A1 was determined kinetically using NADH as competitive inhibitor against NAD1 as reported previously.61,69 Activation parameters a, b (modifying factors of Km and Vmax, respectively), and KA (activating constant) were obtained by global nonlinear fitting of the data from Figure 3, to the Michaelis2Menten equation for mixedtype nonessential activation [Eq. (1)],68 using the software Microcal Origin v. 5.0. t5

 Ks 

½A

11K

V ½S m 

A

b½A

11aK

A

 1½S 

½A

11K



A

b½A

11aK

(1)



A

Determination of Kd for NAD1

ALDH1A1 (20250 lg) was incubated in assay buffer (pH 7.4), titrated with the coenzyme, and the changes in intrinsic fluorescence of the enzyme were followed using an excitation of 287 nm. The emission spectrum in a range from 300 to 400 nm was recorded with the use of an Aminco-Bowman Series 2 spectrofluorometer as previously reported.60 Data were fitted to the Michaelis2Menten equation. Esterase activity

The esterase activity of the enzyme was determined by assaying the rate of p-nitrophenol formation at 400 nm,

A New Specific Modulator of Human ALDH1A1

using a molar extinction coefficient of 14,030 M21 cm21 and incubating the protein in the activity buffer described above, with 0.1 mM p-nitrophenylacetate as the substrate. To determine the concentration of the pnitrophenylacetate stock, a sample was hydrolyzed in 0.1N NaOH, and quantified at 400 nm using an extinction coefficient of 18,300 M21 cm21. Thermal denaturalization assays

Protein (200 mg) was incubated at the indicated temperature in the absence or in the presence of 15 mM tamoxifen, the coenzyme, or the aldehyde, for 5 min. Then, an aliquot (20 mg of protein) was poured into a cuvette containing the assay buffer at 25 C for activity measurement. Statistical analysis

The software GraphPad Prism, version 5.01, was used for Student’s t-test analysis of the data as indicated in the figure legends. RESULTS AND DISCUSSION Docking analysis of ALDHs with tamoxifen

A previous report of the crystal structure of human ALDH2, with ALDA-1 bound (PDB ID: 3INJ), reveled access blocking to the aldehyde binding site by the activator.70 Hence, ALDA-1 activation was attributed to a diminution in active site volume, favoring the number of productive collisions.70 Further analysis revealed that the ALDA-1 heterocyclic hydrophobic segment (N-(1,3-benzodioxol-5-ylmethyl) is stabilized by ALDH-2 hydrophobic amino acids (Val120, Met124, Phe170, Leu173, Phe292, Phe296, Val458, and Phe459) in the funnel of the active site [Fig. 1(B) section (c); see also Ref. 70]. Then, a search for compounds with chemical structures containing a middle aromatic moiety of the adequate size, which might be stabilized in the active site of ALDHs, was conducted, and diclofenac, phloretin, phloridzin, and tamoxifen were selected (Supporting Information Fig. S1). Although the chemical structures of these compounds and ALDA-1 are very different, all the compounds have a hydrophobic segment suitable to be stabilized in the active sites of ALDHs (Supporting Information Fig. S1). These compounds were then assayed by molecular docking for their ability to bind to the catalytic domain of ALDH1A1, ALDH2, and ALDH3A1. Tamoxifen showed the lowest predicted Kd and binding energy with ALDH1A1 among the compounds (Table I), whereas phloretin showed the lowest Kd and energy binding values with ALDH2 (Supporting Information Table S1). Docking analysis indicated that tamoxifen was adequately accommodated in the active sites of ALDH1A1, ALDH2, and ALDH3A1 [Fig. 1(C)

and Supporting Information Fig. S2], but ALDH2 and ALDH3A1 affinities for tamoxifen were two to three orders of magnitude lower (Table I). The docking results also indicated that the phenoxy (middle portion) moiety of tamoxifen may be stabilized by hydrophobic interactions with Met120, Phe170, Leu173, Tyr296, Ile303, Val458, Val459, and Phe465 of ALDH1A1 [Fig. 1(C) section (c)]. Most of these amino acid residues are located in equivalent positions to the hydrophobic residues responsible to stabilize ALDA-1 in the funnel of the ALDH-2 active site [Fig. 1(B) section (c)].70 Although the three enzymes have in essence the same tridimensional structure,23–25 they exhibit more subtle differences in the active site, which may explain their dissimilar tamoxifen binding patterns. The entrance funnel to the aldehyde binding site of ALDH2 is narrower than that of ALDH1A1, which may hinder diffusion to the ALDH2 active site [Supporting Information Fig. S2(A,B)].23,24 Like ALDH1A1, ALDH3A1 seems to have a similar wide entrance to its active site.23,25 However, two Trp residues (residues 56 and 233) are located at the beginning of the aldehyde binding site entrance of ALDH3A1 [Supporting Information Fig. S2(C,D)], which may interfere with the access and binding to the active site. Docking analysis also indicated that the 1,2-diphenylbut-1-enyl (a voluminous aromatic segment) of tamoxifen functioned as an anchor that prevented the compound from reaching further into the active site and to interfere with catalysis [Fig. 1(C) sections (a,b)]; docking of propionaldehyde in the active site in the presence of tamoxifen bound indicated that there was still enough space for binding of the substrate [Fig. 1(C) sections (b,c)]. A similar anchor function for the chloro atoms of the 2,6-dichlorobenzamide moiety of ALDA-1 [Fig. 1(B) sections (a,b)] can be discerned from the activator-ALDH2 crystal.70 Effect of tamoxifen on the kinetics of human ALDH1A1

No effect of diclofenac, phloretin, or phloridzin on the activity of human ALDH1A1, ALDH2, or ALDH3A1 was observed. However, the Vmax of ALDH1A1 was increased by tamoxifen (Fig. 2), whereas no significant effect was observed with human ALDH2 and ALDH3A1; the tamoxifen concentration to reach half-maximal activation (K0.5) of ALDH1A1 was 5.1 6 1.2 mM (n 5 4) [Fig. 2(A)], a concentration 15-fold lower than the EC50 (half-maximal effective concentration) reported for the activation of this enzyme by 2-(benzo[d][1,3]dioxol-5-yl)N-(5,6-dihydro-4H-cyclopenta[c]isoxazol-3-yl)acetamide.54 The magnitude of this activation was similar to that reported for ALDH2 by ALDA-1 (which was less than twofold).39,70 These results showed that tamoxifen was a specific activator of ALDH1A1 as predicted by docking analysis. Kinetic analysis indicated that tamoxifen behaves PROTEINS

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Figure 3 Figure 2 Effect of tamoxifen on the activity of human ALDHs. (A) Titration of the activity of recombinant human ALDH1A1 with tamoxifen. (B) The effect of tamoxifen on the activity of the most studied isozymes of human ALDH. Data are the mean 6 SD of experiments with four independent protein preparations. Vmax values in nmol/min mg: ALDH1 5 190–250; ALDH2 5 590–710; and ALDH3 5 3500–4200. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

as a nonessential mixed-type activator, because it modified the Km for the substrates and the Vmax of the reaction (values of a and b of 0.5 6 0.2 and 1.7 6 0.1, and KA of 4 6 2.3 mM, n 5 4) (Figs. 2 and 3, Table II). The Km for NAD1 diminished by twofold, but no significant effect was observed in the Kd for this substrate, whereas the Km for propionaldehyde slightly increased (Table II), indicating that the activator may indeed interfere with binding, and perhaps entrance, to the active site as predicted by the docking results (Fig. 1). If, as suggested by the docking and kinetic results, tamoxifen is blocking the access to the aldehyde binding site, then both substrates can enter to the active site only through the NAD1 binding site, as also suggested for the activation of ALDH2 by ALDA-1,70 and based on the

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Effect of tamoxifen on the kinetics of human ALDH1A1. Insets show the double reciprocal plots of the hyperbolic curves. ( ), control; the other conditions contained tamoxifen as follows: ( ), 15 lM; ( ), 110 lM; ( ), 115 lM. Plots are representative of the results of experiments with four independent enzyme preparations. Kinetic parameters are summarized in Table II. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

well characterized flexibility of the nicotinamide mononucleotide moiety of NAD1 in ALDHs,23,71,72 that may permit both substrate binding and products release70; nevertheless, the binding profile of the substrates to the Table II Effect of Tamoxifen on the Kinetic Parameters of Human Recombinant ALDH1A1

Km Prop (mM) Km NAD1 (mM) Vmax (nmol/min3mg) Kd NAD1 (lM) a b KA (lM) a

Control

115 mM Tamox

6.5 6 0.7 5.7 6 0.8 217 6 31 8.9 6 0.8

8.7 6 1.4a 2.6 6 0.5b 360 6 55b 8.22 6 0.5 0.5 6 0.2 1.7 6 0.1 4 6 2.3

P < 0.05 P < 0.01 compared with control. Data are the mean 6 SD of experiments with four independent protein preparations. b

A New Specific Modulator of Human ALDH1A1

Figure 4 Effect of tamoxifen on the inhibition pattern of NADH against NAD1. ALDH1A1 was incubated with (D) or without (A) 15 lM tamoxifen, and NADH was used as competitive inhibitor. Concentrations of NADH were: 0 ( ), 12.5 ( ), and 25 ( ) mM. Panels (B) and (E) are the Lineweaver–Burk plots of data from (A) and (D). Data on panels (C) and (F) show the Hanes–Woolf plots of the data to confirm the inhibition patterns. The replots of the slopes from the regressions of data from panels (B) and (E) versus concentration of NADH (data not shown) were used to calculate the Kiq; the mean and SD of the results are presented in Table III. The figure is representative of results of experiments with four independent enzyme preparations. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

enzyme may be altered under these conditions. The kinetic mechanism followed by ALDHs is that of an ordered bi-bi steady-state system; for every kinetic

system, the products show typical inhibition patterns.68 Thus, NADH was used as inhibitor against the substrates to discern whether tamoxifen altered the binding order PROTEINS

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Figure 5 Effect of tamoxifen on the inhibition pattern of NADH against propionaldehyde. ALDH1A1 was incubated with (D) or without (A) 15 lM tamoxifen, and NADH was used as competitive inhibitor. Concentrations of NADH were: 0 ( ), 50 ( ), and 100 ( ) mM. Panels (B) and (E) are the Lineweaver–Burk plots of data from (A) and (D). Data on panels (C) and (F) show the Hanes–Woolf plots of the data to confirm the inhibition patterns. The figure is representative of results of experiments with four independent enzyme preparations. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

of the substrates to the enzyme.68 NADH behaved as a competitive inhibitor against NAD1 under control conditions, whereas it was a mixed-type inhibitor against propionaldehyde, as expected (Figs. 4 and 5).68 In the

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presence of tamoxifen, NADH behaved as a mixed-type inhibitor against NAD1 and as a competitive inhibitor against propionaldehyde (Figs. 4 and 5). These results are indicative of a change in the binding order of the

A New Specific Modulator of Human ALDH1A1

to thermal denaturation was observed when the enzyme was incubated with propionaldehyde, whereas NADH protected by about 50% after 15 min incubation [Fig. 6(A)]. With tamoxifen, the activity of the enzyme was also protected. It is possible that tamoxifen or NADH alone did not fully protect because the NAD1 or aldehyde binding sites were, respectively, empty. Thus, when the combination of NADH and tamoxifen was used, an additive pattern of protection of the activity from thermal denaturation was observed [Fig. 6(A)]. These results suggest that tamoxifen is indeed binding to the enzyme active site. Daidzin is a potent inhibitor of ALDHs73 by interacting with the aldehyde binding site.70,74 Thus, the competition of tamoxifen with the daidzin effect was evaluated. Daidzin inhibited ALDH1A1 activity versus propionaldehyde with a half-maximal inhibitory concentration (IC50) of 30.2 mM, but in the presence of tamoxifen the IC50 increased more than fourfold [Fig. 6(B), Table III), indicating competition between both compounds for the same binding site. Similar results were reported for the interaction of ALDA-1 and daidzin with ALDH2.70 Effect of tamoxifen on the rate-determining step of ALDH1A1

Figure 6 Indirect measurement of the binding of tamoxifen to the active site of ALDH1A1. (A) Protection of the active site of the enzyme to thermal denaturation by the substrates or tamoxifen. The protein was incubated at 45 C for the indicated times in the presence of the following ligands: ( ), protein alone; ( ), 11 mM propionaldehyde; ( ), 115 lM tamoxifen; ( ), 10.5 mM NADH; ( ), 115 lM tamoxifen 1 0.5 mM NADH. Then, the remaining activity was determined as indicated under the Materials and Methods section. Results are representative of experiments with at least three independent protein preparations. (B) Competition of tamoxifen with daidzin for the active site of human ALDH1A1. The enzyme was incubated with ( ) or without ( ) 15 lM tamoxifen in the presence of different concentrations of daidzin, and then the activity was determined as indicated under the Materials and Methods section. Data were fitted to the Hill equation using the software Microcal Origin v. 5.0. Results are the mean 6 SD of experiments with four independent enzyme preparations. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

substrates, propionaldehyde binding first in the presence of the activator. The inhibition profile of NADH against one of the substrates was still competitive in the presence of tamoxifen, and this is consistent with the kinetic mechanism being ordered and not random (see Table IX-6 in Ref. 69). Nevertheless, more experiments are required to establish all the effects of the activator on the kinetic mechanism of the enzyme and to propose a kinetic model. Additional support for the elucidation of the tamoxifen interaction site

To further assess the nature of the tamoxifen binding site, thermostability assays were carried out. No protection

Activation of ALDH1A1 by tamoxifen implies a possible change in the rate-limiting step of the reaction. The reported rate-limiting step of the ALDH1A1 reaction is the release of the reduced coenzyme,75 which can be established by detecting the presence of a pre-steady state NADH “burst” that results from the faster NADH formation but slower release from the active site. ALDH1A1 showed a burst in the presence of tamoxifen (Table III), indicating that the rate-determining step was still located after the formation of NADH. It is well known that ALDHA1 and ALDH2 possess half-of-the-sites reactivity.69,76,77 Then, an alternative explanation for the enzyme activation is the awakening of the otherwise unused active sites in each catalytic cycle by tamoxifen, which may now participate in catalysis. However, the burst magnitude was unchanged in the presence of tamoxifen (i.e., 2 nmol of NADH formed per Table III Effect of Tamoxifen on Different Parameters of ALDH1A1

Activity with propionaldehyde (nmol/min3mg) Daidzin IC50 (mM) Burst magnitude (nmol NADH/nmol enzyme) Esterase activity (nmol/min3mg) Kd NADH (mM)

Control

115 mM Tamox

217 6 31

343 6 42a

30.2 6 6.5 2 6 0.3

139 6 40.7a 2 6 0.5

105 6 15 5.9 6 1.2

82 6 10b 17.6 6 3.9a

a

P < 0.01 P < 0.05 compared with control. Data are the mean 6 SD of experiments with four independent protein preparations. b

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Table IV Effect of Tamoxifen on the Activity of ALDH1A1 with Different Effectors Vmax (nmol/min3mg)

Acetaldehyde Chloroacetaldehyde Mg21, 1 mM

Control

115 mM Tamox

290 6 32 210 6 26a 75 6 23a

430 6 55 275 6 19a 130 6 35a

a P < 0.01 compared with the activity with acetaldehyde in the same column. Data are the mean 6 SD of experiments with four independent protein preparations.

nmol of enzyme; Table III), indicating that the enzyme preserved the half-of-the-sites reactivity. Esterase activity was also assayed to investigate the effect of tamoxifen on the nucleophilicity of Cys 302 (the reactive cysteine of this enzyme). No increase in the esterase activity was detected in the presence of tamoxifen, but a slight decrease in this parameter was observed. An aldehyde with an electron withdrawing group accelerates the deacylation step, and hence it may substantially increase the overall reaction rate as long as the rate-limiting step is the deacylating step.69,77 The activity of ALDH1A1 with chloroacetaldehyde was 70% of that achieved with acetaldehyde, whereas in the presence of tamoxifen the activity with the chloro-substituted aldehyde was 78% of that observed with acetaldehyde (Table IV). These results indicated that the ratedetermining step of the enzyme in the presence of tamoxifen is not the deacylation step. Mg21 has differential effects on the deacylation and reduced coenzyme release steps, activating the first, whereas inhibiting the second step.69,77–79 Addition of Mg21 inhibited the activity of ALDH1A1 by 65% with and without tamoxifen (Table IV), clearly indicating that the rate-limiting step was still located at the coenzyme releasing step. To further support the last conclusion, the dissociation constant of the reduced coenzyme (Kiq) was determined. The Kiq value increased threefold in the presence of tamoxifen (Table III). Because coenzyme dissociation is the rate-determining step for the ALDHA1A reaction, any modification in the NADH affinity will affect the rate of the global reaction. In conclusion, the docking and kinetic data (including mixed-type activation, modification of the order of binding of the substrates, thermostability, and competition with daidzin for binding) suggested that tamoxifen binds to the aldehyde active site. Although tamoxifen binding may slightly decrease the active site volume, activation of ALDH1A1 by tamoxifen was more related to a diminution in the affinity for NADH, favoring this product release and thus increasing the global rate of the reaction. The elucidation of the mechanism of activation of ALDH1A1 by tamoxifen reported in this work may contribute to the improvement of the development of

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A New Specific Modulator of Human ALDH1A1

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