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Flavonoids: True or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase
Accepted Manuscript Flavonoids: true or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase Denis Tritsch, Catherine Zinglé, Michel Rohmer, Catherine GrosdemangeBilliard PII: DOI: Reference:
S0045-2068(15)00017-6 http://dx.doi.org/10.1016/j.bioorg.2015.02.008 YBIOO 1794
To appear in:
Bioorganic Chemistry
Received Date:
20 January 2015
Please cite this article as: D. Tritsch, C. Zinglé, M. Rohmer, C. Grosdemange-Billiard, Flavonoids: true or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.02.008
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Flavonoids: true or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase.
Denis TRITSCH*, Catherine ZINGLÉ, Michel ROHMER and Catherine GROSDEMANGEBILLIARD*
Université de Strasbourg/CNRS, Strasbourg, UMR 7177, Institut Le Bel, 4 rue Blaise Pascal, 67081 Strasbourg, France * Corresponding authors: Tel.: +33 368 851 353 (D.T.); Tel.: +33 368 851 349; E-mail addresses: [email protected] (D. Tritsch), [email protected] (C. Grosdemange-Billiard)
Abstract: Flavonoids, due to their physical and chemical properties (among them hydrophobicity and metal chelation abilities), are potential inhibitors of the 1-deoxyxylulose 5-phosphate reductoisomerase and most of the tested flavonoids effectively inhibited its activity with encouraging IC50 values in the micromolar range. The addition of 0.01% Triton X100 in the assays led however, to a dramatic decrease of the inhibition revealing that a non-specific inhibition probably takes place. Our study highlights the possibility of erroneous conclusions regarding the inhibition of enzymes by flavonoids that are able to produce aggregates in micromolar range. Therefore, the addition of a detergent in the assays prevents possible false positive hits in high throughput screenings.
Keywords: flavonoids, 1-deoxyxylulose 5-phosphate reductoisomerase, promiscuous inhibition, 2-C-methyl-D-erythritol 4-phosphate pathway Abbreviations MEP: 2-C-methyl-D-erythritol 4-phosphate DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase H-DXR: His-tagged DXR DXP: 1-deoxy-D-xylulose 5-phosphate
1
1. Introduction Flavonoids are secondary plant metabolites essentially known for their antioxidant properties. They have also antiviral, antiprotozoal and interestingly antimicrobial activities [1-3], which are well reported in the PubChem BioAssays database [4]. Flavonoids inhibit many unrelated enzymes (see Table S1 for an extensive list of enzymes inhibited by these compounds). They can bind to enzymes according to different mechanisms owing to their multiple binding possibilities: hydrogen bonding, hydrophobic interactions, metal chelation and ππ-stacking, with a tendency to occupy hydrophobic pockets [5]. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, involved in the biosynthesis of isoprenoids, constitutes a valuable target to conceive new antimicrobial drugs [6]. This pathway is notably present in some extremely virulent pathogens such as Mycobacterium tuberculosis, the causative agent of tuberculosis, and Plasmodium falciparum, the parasite responsible for malaria [7,8]. The inhibition of this pathway is particularly attractive as it is absent in humans. The inhibition of 1-deoxyxylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the pathway, is the most studied since the natural product fosmidomycin [9,10] was found to be an efficient inhibitor of this enzyme [11]. Unfortunately, bacteria become rapidly resistant, restricting its use in antibiotic therapies [12,13]. The syntheses of multiple fosmidomycin derivatives to improve its pharmacological properties gave rather disappointing results. Among the tested analogues, only few have similar or slightly better inhibition potency [14-19]. With the aim to find new hits, we tested the ability of flavonoids to inhibit the activity of the E. coli DXR (Figure 1). Some considerations motivated this study. Firstly, their metal chelation potential lets envisage that they might chelate the Mg2+ cation present in the DXR active site. Flavonoids containing a catechol group were accordingly selected to optimize the number of chelation sites. Indeed, catechols form stable complexes with the magnesium dication Mg2+, a hard metal ion [20]. In addition, flavonoids may act as potential efficient inhibitors as the DXR has hydrophobic pockets situated close to the active site where they might interact and interfere with the enzymatic activity [21]. Finally, considering their structures, a binding in the NADPH recognition site is also conceivable. Analysing a set of flavonoids with different structure should thus allow a structure-activity relationship study.
2
Figure 1: Flavonoids tested on E. coli H-DXR
An aspect is however often neglected in enzyme inhibition studies with flavonoids, the possibility of the rather hydrophobic molecules to give aggregates in aqueous solution. Their formation depends not only on the flavonoid structures but also on their concentration and the nature of the buffer [22]. These aggregates are capable of inhibiting enzyme in a non-specific manner [23-26]. The inhibition seems to occur via a partial unfolding of the enzymes once they are bound to such aggregates [27]. Such inhibitors, also called aggregating or promiscuous inhibitors, are considered as “false positives” in a high throughput screening, and are unsuitable as lead compounds [28]. As some flavonoids have a promising potential to inhibit the bacterial DXR, the influence of selected flavonoids was analysed on the activity of His-tagged DXR of Escherichia coli (HDXR). Discrimination between specific and non-specific inhibition by these flavonoids was attempted. A basic method is to compare the inhibition induced by the compounds in the
3
presence and the absence of a non-ionic detergent [29,30]. A non-specific inhibition will be established when the detergent leads to a dramatic decrease of the inhibition level. 2. Results Suspecting that flavonoids could be slow-binding inhibitors like fosmidomycin, we tested the influence of preincubation of quercetin on the DXR inhibition [31,32]. Clearly, a significant increase of the inhibition was observed when flavonoids were preincubated with DXR in the absence of NADPH. At a 20 µM concentration, the inhibition increased from 30% to 81% when the flavonoid was incubated during 2 min with DXP and H-DXR. Accordingly, the enzyme assays were all performed after a preincubation of the potential inhibitors with HDXR. The inhibition of H-DXR by flavonoids is determined by the measure of IC50 or by calculating the percentage of inhibition. (Table 1) Among the tested flavonoids those possessing a catechol group on ring A or B inhibited the enzyme at a low micromolar concentration. Morin with a resorcinol group on ring B as well the flavan-3-ols (+)-catechin and (-)-epicatechin, while bearing a catechol group were much less efficient. Rutin, a 3-O glycosylated quercetin derivative did not inhibit H-DXR. No Triton Flavonoids
With Triton
IC50 (µM)
% of inhibition
% of inhibition
Luteolin
3.4
-
37 a
Quercetin
1.9
-
31 a
-
7b
Rutin Morin
b
ND ND
-
56
Myricetin #
1.5
-
27 a
Baicalein
2.2
-
<5a
Fisetin
3.5
-
21 a
(+)-Catechin
-
40 b
ND
(-)-Epicatechin
-
52 b
ND
Catechol Fosmidomycin*
-
7
0.042
-
b
ND -
Reactions were conducted as described in Experimental. When feasible, IC50 were determined, otherwise the percentages of inhibition at an inhibitor concentration of a 20 µM or b 80 µM are given. (ND not determined). * The IC50 of fosmidomycin was determined as described in [31]. # Illustration of influence of Triton X100 on the inhibition of H-DXR with myricetin (Figure S1).
4
Table 1: Influence of the presence of 0.01% Triton X100 on the inhibition of H-DXR with flavonoids and related compounds
The presence of detergent led to a significant decrease of the inhibition potency (Table 1). It was obvious with baicalein where the inhibition was almost non-existent. When the concentration of flavonoids in the reaction medium varied, no clear dose-dependent inhibition was observed. For example with quercetin, the inhibition remained constant at a level of about 20-25% between 1 to 20 µM flavonoid concentrations. This observation might indicate that a specific inhibition, to some extent, by several flavonoids could not be excluded. As the compounds are susceptible to target the coenzyme binding site, we determined the influence of NADPH on the inhibition level (Table 2). Even at a saturating concentration of NADPH, the protection was only partial, suggesting that flavonoids probably do not bind to the NADPH binding site. Flavonoids
concentration (µM)
% inhibition
% inhibition
No NADPH
160 µM NADPH
Luteolin
6
67
38
Quercetin
4
77
44
Myricetin
4
82
47
Baicalein
4
75
46
Fisetin
6
67
41
The enzyme was pre-incubated during 2 min at 37°C with the inhibitors in the presence or in the absence of NADPH (160 µM final concentration). The enzymatic reaction was initiated by addition of DXP (0.5 mM final concentration) followed by that of NADPH in the first case and by the addition of DXP in the second case.
Table 2: Protection of H-DXR against flavonoid inhibition by NADPH
As the flavonoids did not seem to fit in the cofactor binding site, we studied the influence of the concentration of DXP on the inhibition of the enzyme by myricetin. Our kinetic studies revealed that myricetin behaves as a pure uncompetitive inhibitor of H-DXR versus DXP (Figure 2). The replots (apparent 1/Vmax and 1/Km vs myricetin concentration, see Figure S2) were linear allowing the determination of a Ki value of 1.1 µM [33]. DXR is described to proceed via a sequential ordered Bi-Bi mechanism with NADPH binding first to the enzyme 5
[32]. The uncompetitive character of the inhibition seemed to indicate that DXP is able to bind to the enzyme in the absence of NADPH. This apparent discrepancy implies that, in the presence of NADPH and DXP, the cofactor will bind faster to the enzyme than DXP. 100
1/V0 (min mM-1)
90 80 70 60 50 40 30 20 10 0 -5
0
5
1/[DXP]
10
15
(mM-1)
Reactions were conducted as described in Material and Methods. The enzyme was pre-incubated with DXP (concentration between 0.096 and 0.48 mM) and myricetin at a concentration of 0 (), 0.5 (), 1 () and 2 () µM. The reaction was initiated with NADPH at a final concentration of 160 µM.
Figure 2: Inhibition of H-DXR by myricetin.
3. Discussion Flavonoids were described to inhibit numerous unrelated enzymes. No general rules could be provided for the binding of these molecules to the enzymes except the predominance of hydrophobic interactions between the flavonoids and the enzymes [5]. We tested the influence of some flavonoids on the enzymatic activity of H-DXR with aim to find new inhibitors. We compared a variety of flavonoids possessing similar core structures but differing in single structural elements, such as the presence or absence of a double bond between C-2 and C-3 and the hydroxylation pattern. As the hydroxyl groups may generate hydrogen bonds with amino acids or peptide bonds, the presence or the absence of a hydroxyl group on the carbon skeleton can modulate the inhibitory effect of flavonoids. Half of the tested flavonoids inhibited the H-DXR in a dose-dependent manner in the low micromolar range. The possibility of a non-specific inhibition due to colloidal aggregation of flavonoids is often neglected and a validation by additional proofs than IC50 values to identify them as specific 6
inhibitors is required. In some cases, the specific binding was validated by resolving the crystal structure of the flavonoid-enzyme complex, providing an insight into the binding mode (see Table S2). Other methods, easier to carry out, based on kinetic properties were also proposed as, for instance, the comparison of the inhibition in the presence or the absence of a non-ionic detergent in the reaction medium. It was assumed that the detergent would disperse the aggregates inducing a dramatic decrease of the inhibition in the case of a promiscuous inhibition. In contrast, a weak increase of the IC50 would mean that the inhibition is probably specific. As Triton X100, at least until a concentration of 0.1%, had no effect on the activity of H-DXR, we could compare the inhibition induced by flavonoids in presence and absence of the detergent as proposed earlier [29,30]. A noticeable decrease of inhibition was effectively observed in presence of the detergent. Thus, according to the criteria established by Shoichet and co-workers, the tested flavonoids inhibit probably the enzyme via a non-specific mechanism. The method itself seems not questionable as it was already successfully used to prove the specificity of inhibition of some enzymes [34-39]. The phenomenon was especially noticeable for baicalein since no inhibition was observed in the presence of Triton X100. We could exclude that the absence of inhibition was due to the sequestering of the flavonoid by Triton X100 molecules, as in the case of the human 12-lipoxygenase, baicalein inhibited even in the presence of the detergent [34]. Two other features support a non-specific inhibition of H-DXR by flavonoids. The fact that a pre-incubation of the enzyme with the flavonoids led to an increase of the inhibition, is a necessary though insufficient condition. The lack of protection of NADPH and the uncompetitive inhibition versus DXP are also in favour of an aggregation-based inhibition. Indeed a specific inhibitor should rather be competitive versus the substrate(s). Morin and the flavan-3-ols were ineffective to inhibit H-DXR. In a first approach, these results could be explained for morin, by the presence of a resorcinol instead a catechol group on ring B but also by the failure to give aggregates at the tested concentration [22]. Concerning the tested flavan-3-ols, the loss of the nearly perfect coplanarity existing between ring B and the benzo-γ-pyrone moiety due to the lack of the 2,3 double bond could be responsible for the binding affinity diminution but, as for morin, no formation of aggregates was observed in solution [22]. Moreover, the fact that the tested flavonoids did not inhibit the growth of E. coli (results not shown) strengthens the postulated non-specific inhibition of those compounds.
4. Conclusion 7
The possibility of inhibition by aggregates is often neglected in particular if hydrophobic molecules with very low water solubility are tested. Our results showed that the flavonoids inhibiting the H-DXR can be considered, with high probability, as promiscuous inhibitors. The recently introduced notion of Pan Assay Interference Compounds or PAINS has to be considered in high throughput screening to avoid false positive inhibitors [40]. While described as aggregators, flavonoids, owing to their valuable binding properties, are not to be neglected but particular conditions of assay have to be investigated to be sure that these compounds could be considered as new hits. The addition of detergent in the assays, for instance, would be helpful to prevent false positives in high throughput screening [41,42] and especially when the IC50, in the absence of detergent, are in the micromolar range [43]. It is obvious that affinities in the nanomolar range and high selectivity could restrain the problem of aggregation-based inhibition.
5. Experimental 5.1. Material NADPH was purchased from Sigma-Aldrich. Flavonoids (purity ≥ 95%) were purchased from Sigma-Aldrich, Carl Roth GmbH (Karlsruhe, Germany) or Extrasynthese (Genay, France). They were used without further purification. DXP was synthesized according to Meyer et al.[44]. The cloning and purification of His-tagged DXR (H-DXR) was previously described [31].
5.2. H-DXR activity [31] The assays were performed at 37°C in a 50 mM Tris/HCl buffer pH 7.5 containing 3 mM MgCl2 and 2 mM DTT. The volume was 500 µL. The enzyme was pre-incubated during 2 min with DXP. The enzymatic reaction was initiated by addition of NADPH. The concentrations of DXP and NADPH were 480 µM and 160 µM respectively. The decrease of absorbance at 340 nm due to NADPH oxidation was monitored with an Uvikon 933 UV-Vis spectrophotometer to determine the initial rates. The retained values were the average of at least two measurements. The relative average deviation must be lower than 4 %. The protein concentration was determined by the Bradford method using the Bio-Rad protein assay and bovine serum albumin as the standard [45].
8
5.3. Inhibition studies The DXR inhibition by the flavonoids was quantified by determining the IC50 values. They were obtained by plotting the percentage of residual activity versus the log of inhibitor concentration. In some cases, due the limited solubility of the compounds and/or the low inhibition potential, only the percentage of inhibition at given concentrations (20 or 80 µM) was given. H-DXR was pre-incubated during 2 min in the presence of the inhibitors at different concentrations and DXP (480 µM). NADPH (160 µM final concentration) was then added to measure the residual activity. The flavonoids are poorly soluble in water. Flavonoids solutions at different concentrations were prepared in DMSO by serial dilutions from 100 mM stock solutions. Aliquots of the solutions were added in the assays so that the concentration of DMSO did not exceed 0.4% (v/v). Triton X100 (0.01%) was included in the reaction medium to determine its influence on the inhibition capacity of the tested flavonoids. The inhibition mechanism of myricetin was studied by pre-incubating H-DXR with DXP at varied concentrations (96 to 480 µM) and fixed concentrations of myricetin (0.5, 1 and 2 µM). The enzymatic reaction was initiated by NADPH (final concentration 160 µM). To test the influence of NADPH on the inhibition, the enzyme was pre-incubated with the inhibitors and NADPH (160 µM) during 2 min. DXP (480 µM final concentration) was added to initiate the reaction and determine the residual activity. The H-DXR inhibitor fosmidomycin was used as a positive reference. It was tested as described in [31]. An IC50 value of 42 nM was measured. As the value was near that obtained in earlier studies, we considered that the enzyme was functionally equivalent to that used previously.
5.4. Bacterial growth inhibition The antimicrobial activity of each inhibitor of H-DXR was tested by the paper disc diffusion method on Escherichia coli XL1 Blue [31]. Paper discs were impregnated with 400 nanomoles of inhibitors. Fosmidomycin (10 nanomoles) was the positive reference.
Conflicts of interest The authors declare no conflicts of interest.
Acknowledgements 9
C. Zinglé was a recipient of a grant from the ‘Ministère de la Recherche’. This work was supported by a grant from the “Agence Nationale de la Recherche” (Grant Nb ANR-06BLAN-0291-01).
Supplementary material Table S1: List of enzyme inhibited by flavonoids Table S2: List of X-ray structures of (enzyme, flavonoids) complexes Figure S1: Influence of Triton X100 on the inhibition of H-DXR with myricetin Figure S2: Replot of 1/Km apparent vs myricetin concentration and replot of 1/Vmax apparent vs myricetin concentration
References
[1]
B.H. Havsteen, The biochemistry and medical significance of the flavonoids, Pharmacol. Ther. 96 (2002) 67-202.
[2]
T.P.T. Cushnie, A.J. Lamb, Antimicrobial activity of flavonoids, Int. J. Antimicrob. Agents 26 (2005) 343-356.
[3]
T.P.T. Cushnie, A.J. Lamb, Recent advances in understanding the antibacterial properties of flavonoids, Int. J. Antimicrob. Agents 38 (2011) 99-107.
[4]
National Center for Biotechnology Information - PubChem BioAssay database http://wwwncbinlmnihgov/
[5]
O. Dangles , C. Dufour, Flavonoid-protein interactions in Flavonoids: Chemistry, Biochemistry and Applications Edited by Øyvind M Andersen and Kenneth R Markham, CRC Press (2005) pp 443-469
[6]
M. Rohmer, The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants, Nat. Prod. Rep. 16 (1999) 565-574.
[7]
H. Jomaa, J. Wiesner, S. Sanderbrand, B. Altincicek, C. Weidemeyer, M. Hintz, I. Türbachova, M. Eberl, J. Zeidler, H.K. Lichtenthaler, D. Soldati, E. Beck, Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs, Science 285 (1999) 1573-1576.
[8]
M. Rohmer, C. Grosdemange-Billiard, M. Seemann, D. Tritsch, Isoprenoid biosynthesis as a novel target for antibacterial and antiparasitic drugs, Curr. Opin. Invest. Drugs 5 (2004) 154-162.
10
[9]
M. Okuhara, Y. Kuroda, T. Goto, M. Okamoto, H. Terano, M. Kohsaka, H. Aoki, H. Imanaka, Studies on new phosphonic acid antibiotics. III. Isolation and characterization of FR-31564, FR-32863 and FR-33289, J. Antibiot. 33 (1980) 24-28.
[10]
Y. Shigi, Inhibition of bacterial isoprenoid synthesis by fosmidomycin, a phosphonic acid-containing antibiotic, J. Antimicrob. Chemother. 24 (1989) 131-145.
[11]
T. Kuzuyama, T. Shimizu, S. Takahashi, H. Seto, Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis, Tetrahedron Lett. 39 (1998) 7913-7916.
[12]
A.S. Messiaen, T. Verbrugghen, C. Declerck, R. Ortmann, M. Schlitzer, H. Nelis, S. Van Calenbergh, T. Coenye, Resistance of the Burkholderia cepacia complex to fosmidomycin and fosmidomycin derivatives, Int. J. Antimicrob. Agents 38 (2011) 261-264.
[13]
A. Hemmerlin, D. Tritsch, P. Hammann, M. Rohmer, T.J. Bach, Profiling of defense responses in Escherichia coli treated with fosmidomycin, Biochimie 99 (2014) 54-62.
[14]
M. Andaloussi, L.M. Henriksson, A. Wieckowska, M. Lindh, C. Björkelid, A.M. Larsson, S. Suresh, H. Iyer, B.R. Srinivasa, T. Bergfors, T. Unge, S.L. Mowbray, M. Larhed, T.A. Jones, A. Karlén, Design, synthesis, and X-ray crystallographic studies of α-aryl substituted fosmidomycin analogues as inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, J. Med. Chem. 54 (2011) 4964-4976.
[15]
C.T. Behrendt, A. Kunfermann, V. Illarionova, A. Matheeussen, M.K. Pein, T. Gräwert, J. Kaiser, A. Bacher, W. Eisenreich, B. Illarionov, M. Fischer, L. Maes, M. Groll, T. Kurz, Reverse fosmidomycin derivatives against the antimalarial drug target ispC (Dxr), J. Med. Chem. 54 (2011) 6796-6802.
[16]
C. Zinglé, L. Kuntz, D. Tritsch, C. Grosdemange-Billiard, M. Rohmer, Isoprenoid biosynthesis via the methylerythritol phosphate pathway: structural variations around phosphonate anchor and spacer of fosmidomycin, a potent inhibitor of deoxyxylulose phosphate reductoisomerase, J. Org. Chem. 75 (2010) 3203-3207.
[17]
T. Kurz, D. Geffken, C. Wackendorff, Hydroxyurea analogues of fosmidomycin, Z. Naturforsch 58b (2003) 106-110.
[18]
M. Courtois, Z. Mincheva, F. Andreu, M. Rideau, M.C. Viaud-Massuard, Synthesis and biological evaluation with plant cells of new fosmidomycin analogues containing a benzoxazolone or oxazolopyridinone ring, J. Enzyme Inhib. Med. Chem. 19 (2004) 559-565.
11
[19]
C. Zinglé, L. Kuntz, D. Tritsch, C. Grosdemange-Billiard, M. Rohmer, Modifications around the hydroxamic acid chelating group of fosmidomycin, an inhibitor of the metalloenzyme 1-deoxyxylulose 5phosphate reductoisomerase (DXR), Bioorg. Med. Chem. Lett. 22 (2012) 6563-6567.
[20]
V.T. Athavale, L.H. Prabhu, D.G. Vartak, Solution stability constants of some metal complexes of derivatives of catechol, J. Inorg. Nucl. Chem. 28 (1966) 1237-1249.
[21]
L. Deng, K. Endo, M. Kato, G. Cheng, S. Yajima, Y. Song, Structures of 1deoxy-D-xylulose 5-phosphate reductoisomerase/lipophilic phosphonate complexes, ACS Med. Chem. Lett. 2 (2011) 165-170.
[22]
L. Pohjala, P. Tammela, Aggregating behavior of phenolic compounds-a source of false bioassay results, Molecules 17 (2012) 10774-10790.
[23]
S.L. McGovern, E. Caselli, N. Grigorieff, B.K. Shoichet, A common mechanism underlying promiscuous inhibitors from virtual and highthroughput screening, J. Med. Chem. 45 (2002) 1712-1722.
[24]
S.L. McGovern, B.K. Shoichet, Kinase inhibitors: not just for kinases anymore, J. Med. Chem. 46 (2003) 1478-1483.
[25]
S.L. McGovern, B.T. Helfand, B. Feng, B.K. Shoichet, A specific mechanism of nonspecific inhibition, J. Med. Chem. 46 (2003) 4265-4272.
[26]
J. Seidler, S.L. McGovern, T.N. Doman, B.K. Shoichet, Identification and prediction of promiscuous aggregating inhibitors among known drugs, J. Med. Chem. 46 (2003) 4477-4486.
[27]
K.E.D. Coan, D.A. Maltby, A.L. Burlingame, B.K. Shoichet, Promiscuous aggregate-based inhibitors promote enzyme unfolding, J. Med. Chem. 52 (2009) 2067-2075.
[28]
A. Whitty, Growing PAINS in academic drug discovery, Future Med. Chem. 3 (2011) 797-801.
[29]
B.Y. Feng, B.K. Shoichet, A detergent-based assay for the detection of promiscuous inhibitors, Nat. Protoc. 1 (2006) 550-553.
[30]
A.J. Ryan, N.M. Gray, P.N. Lowe, C. Chung, Effect of detergent on “promiscuous” inhibitors, J. Med. Chem. 46 (2003) 3448-3451.
[31]
L. Kuntz, D. Tritsch, C. Grosdemange-Billiard, A. Hemmerlin, A. Willem, T.J. Bach, M. Rohmer, Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase, Biochem. J. 386 (2005) 127135.
12
[32]
A.T. Koppisch, D.T. Fox, B.S. Blagg, C.D. Poulter, E. coli MEP synthase: steady-state kinetic analysis and substrate binding, Biochemistry 41 (2002) 236-243.
[33]
I.H. Segel, Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems, Wiley-Interscience, NewYork; 1975: 161226.
[34]
J.D. Deschamps, V.A. Kenyon, T.R. Holman, Baicalein is a potent in vitro inhibitor against both reticulocyte 15-human and platelet 12-human lipoxygenases, Bioorg. Med. Chem. 14 (2006) 4295-4301.
[35]
R.F. Freitas, I.M. Prokopczyk, A. Zottis, G. Oliva, A.D. Andricopulo, M.T.S. Trevisan, W. Vilegas, M.G.V. Silva, C.A. Montanari, Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors, Bioorg. Med. Chem. 17 (2009) 2476-2482.
[36]
M. Brunskole, K. Zorko, V. Kerbler, S. Martens, J. Stojan, S. Gobec, T. Lanisnik Rizner, Trihydroxynaphthalene reductase of Curvularia lunata - a target for flavonoid action?, Chem. Biol. Interact. 178 (2009) 259-267.
[37]
S. Arioka, M. Sakagami, R. Uematsu, H. Yamaguchi, H. Togame, H. Takemoto, H. Hinou, S.-I. Nishimura, Potent inhibitor scaffold against Trypanosoma cruzi trans-sialidase, Bioorg. Med. Chem. 18 (2010) 16331640.
[38]
T. Tarragó, N. Kichik, B. Claasen, R. Prades, M. Teixidó, E. Giralt, Baicalein, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor, Bioorg. Med. Chem. 16 (2008) 7516-7524.
[39]
D. Duan, A. K. Doak, L. Nedyalkova, B. K. Shoichet, Colloidal aggregation and the in vitro activity of traditional chinese medicines, ACS Chem. Biol (2015) in press
[40]
J.B. Baell, G.A. Holloway, New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays, J. Med. Chem. 53 (2010) 2719-2740.
[41]
A. Fallarero, L. Hanski, P. Vuorela, How to translate a bioassay into a screening assay for natural products: General considerations and implementation of antimicrobial screens, Planta Med. 80 (2014) 1182-1199.
[42]
X. Xie, L. Sun, Z.Y. Pesetto, Y. Zhao, Z. Zang, L. Zhong, M. Wu, Q. Su, X. Gao, W. Zan, Y. Sun, Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators, PLoS One 8 (2013) e78042.
13
[43]
C. Zinglé, D. Tritsch, C. Grosdemange-Billiard, M. Rohmer, Catecholrhodanine derivatives: Specific and promiscuous inhibitors of Escherichia coli deoxyxylulose phosphate reductoisomerase (DXR), Bioorg. Med. Chem. 22 (2014) 3713-3719.
[44]
O. Meyer, J.-F. Hoeffler, C. Grosdemange-Billiard, M. Rohmer Practical synthesis of 1-deoxy-D-xylulose and 1-deoxy-D-xylulose 5-phosphate allowing deuterium labelling, Tetrahedron 60 (2004) 12153-12262.
[45]
M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle protein-dye binding, Anal. Biochem. 72 (1976) 248-254.
14
Tables Table 1
Influence of the presence of 0.01% Triton X100 on the inhibition of H-DXR with flavonoids and related compounds
No Triton Flavonoids
With Triton
IC50 (µM)
% of inhibition
% of inhibition
Luteolin
3.4
-
37 a
Quercetin
1.9
-
31 a
-
7b
Rutin Morin
b
ND
-
56
Myricetin
1.5
-
27 a
Baicalein
2.2
-
<5a
Fisetin
3.5
-
21 a
(+)-Catechin
-
40 b
ND
(-)-Epicatechin
-
52 b
ND
Catechol
-
7b
ND
0.042
-
-
Fosmidomycin*
ND
Reactions were conducted as described in Experimental. When feasible, IC50 were determined, otherwise the percentages of inhibition at an inhibitor concentration of a 20 µM or b 80 µM are given. (ND not determined). * The IC50 of fosmidomycin was determined as described in [31].
15
Table 2
Protection of H-DXR by NADPH against flavonoid inhibition
Flavonoids
concentration (µM)
% inhibition
% inhibition
No NADPH
160 µM NADPH
Luteolin
6
67
38
Quercetin
4
77
44
Myricetin
4
82
47
Baicalein
4
75
46
Fisetin
6
67
41
The enzyme was pre-incubated during 2 min at 37°C with the inhibitors in the presence or in the absence of NADPH (160 µM final concentration). The enzymatic reaction was initiated by addition of DXP (0.5 mM final concentration) followed by that of NADPH in the first case and by the addition of DXP in the second case.
16
Figures
Figure 1: Flavonoids tested on His-tagged DXR of E. coli
17
100
1/V0 (min mM-1)
90 80 70 60 50 40 30 20 10 0 -5
0
5
10
15
1/[DXP] (mM-1)
Figure 2: Inhibition of H-DXR by myricetin. Reactions were conducted as described in Material and Methods. The enzyme was preincubated with DXP (concentration between 0.096 and 0.48 mM) and myricetin at a concentration of 0 (), 0.5 (), 1 () and 2 () µM. The reaction was initiated with NADPH at a final concentration of 160 µM.
18
Graphical abstract
19
Highlights Possible erroneous conclusions regarding the inhibition of enzymes by flavonoids Hydrophobic molecules can generate aggregates responsible of enzyme inhibition Inhibition studies with flavonoids require additional testing with non-ionic detergent Flavonoids inhibit deoxyxylulose phosphate reductoisomerase via a non-specific way
20
S0045-2068(15)00017-6 http://dx.doi.org/10.1016/j.bioorg.2015.02.008 YBIOO 1794
To appear in:
Bioorganic Chemistry
Received Date:
20 January 2015
Please cite this article as: D. Tritsch, C. Zinglé, M. Rohmer, C. Grosdemange-Billiard, Flavonoids: true or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.02.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Flavonoids: true or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase.
Denis TRITSCH*, Catherine ZINGLÉ, Michel ROHMER and Catherine GROSDEMANGEBILLIARD*
Université de Strasbourg/CNRS, Strasbourg, UMR 7177, Institut Le Bel, 4 rue Blaise Pascal, 67081 Strasbourg, France * Corresponding authors: Tel.: +33 368 851 353 (D.T.); Tel.: +33 368 851 349; E-mail addresses: [email protected] (D. Tritsch), [email protected] (C. Grosdemange-Billiard)
Abstract: Flavonoids, due to their physical and chemical properties (among them hydrophobicity and metal chelation abilities), are potential inhibitors of the 1-deoxyxylulose 5-phosphate reductoisomerase and most of the tested flavonoids effectively inhibited its activity with encouraging IC50 values in the micromolar range. The addition of 0.01% Triton X100 in the assays led however, to a dramatic decrease of the inhibition revealing that a non-specific inhibition probably takes place. Our study highlights the possibility of erroneous conclusions regarding the inhibition of enzymes by flavonoids that are able to produce aggregates in micromolar range. Therefore, the addition of a detergent in the assays prevents possible false positive hits in high throughput screenings.
Keywords: flavonoids, 1-deoxyxylulose 5-phosphate reductoisomerase, promiscuous inhibition, 2-C-methyl-D-erythritol 4-phosphate pathway Abbreviations MEP: 2-C-methyl-D-erythritol 4-phosphate DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase H-DXR: His-tagged DXR DXP: 1-deoxy-D-xylulose 5-phosphate
1
1. Introduction Flavonoids are secondary plant metabolites essentially known for their antioxidant properties. They have also antiviral, antiprotozoal and interestingly antimicrobial activities [1-3], which are well reported in the PubChem BioAssays database [4]. Flavonoids inhibit many unrelated enzymes (see Table S1 for an extensive list of enzymes inhibited by these compounds). They can bind to enzymes according to different mechanisms owing to their multiple binding possibilities: hydrogen bonding, hydrophobic interactions, metal chelation and ππ-stacking, with a tendency to occupy hydrophobic pockets [5]. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, involved in the biosynthesis of isoprenoids, constitutes a valuable target to conceive new antimicrobial drugs [6]. This pathway is notably present in some extremely virulent pathogens such as Mycobacterium tuberculosis, the causative agent of tuberculosis, and Plasmodium falciparum, the parasite responsible for malaria [7,8]. The inhibition of this pathway is particularly attractive as it is absent in humans. The inhibition of 1-deoxyxylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the pathway, is the most studied since the natural product fosmidomycin [9,10] was found to be an efficient inhibitor of this enzyme [11]. Unfortunately, bacteria become rapidly resistant, restricting its use in antibiotic therapies [12,13]. The syntheses of multiple fosmidomycin derivatives to improve its pharmacological properties gave rather disappointing results. Among the tested analogues, only few have similar or slightly better inhibition potency [14-19]. With the aim to find new hits, we tested the ability of flavonoids to inhibit the activity of the E. coli DXR (Figure 1). Some considerations motivated this study. Firstly, their metal chelation potential lets envisage that they might chelate the Mg2+ cation present in the DXR active site. Flavonoids containing a catechol group were accordingly selected to optimize the number of chelation sites. Indeed, catechols form stable complexes with the magnesium dication Mg2+, a hard metal ion [20]. In addition, flavonoids may act as potential efficient inhibitors as the DXR has hydrophobic pockets situated close to the active site where they might interact and interfere with the enzymatic activity [21]. Finally, considering their structures, a binding in the NADPH recognition site is also conceivable. Analysing a set of flavonoids with different structure should thus allow a structure-activity relationship study.
2
Figure 1: Flavonoids tested on E. coli H-DXR
An aspect is however often neglected in enzyme inhibition studies with flavonoids, the possibility of the rather hydrophobic molecules to give aggregates in aqueous solution. Their formation depends not only on the flavonoid structures but also on their concentration and the nature of the buffer [22]. These aggregates are capable of inhibiting enzyme in a non-specific manner [23-26]. The inhibition seems to occur via a partial unfolding of the enzymes once they are bound to such aggregates [27]. Such inhibitors, also called aggregating or promiscuous inhibitors, are considered as “false positives” in a high throughput screening, and are unsuitable as lead compounds [28]. As some flavonoids have a promising potential to inhibit the bacterial DXR, the influence of selected flavonoids was analysed on the activity of His-tagged DXR of Escherichia coli (HDXR). Discrimination between specific and non-specific inhibition by these flavonoids was attempted. A basic method is to compare the inhibition induced by the compounds in the
3
presence and the absence of a non-ionic detergent [29,30]. A non-specific inhibition will be established when the detergent leads to a dramatic decrease of the inhibition level. 2. Results Suspecting that flavonoids could be slow-binding inhibitors like fosmidomycin, we tested the influence of preincubation of quercetin on the DXR inhibition [31,32]. Clearly, a significant increase of the inhibition was observed when flavonoids were preincubated with DXR in the absence of NADPH. At a 20 µM concentration, the inhibition increased from 30% to 81% when the flavonoid was incubated during 2 min with DXP and H-DXR. Accordingly, the enzyme assays were all performed after a preincubation of the potential inhibitors with HDXR. The inhibition of H-DXR by flavonoids is determined by the measure of IC50 or by calculating the percentage of inhibition. (Table 1) Among the tested flavonoids those possessing a catechol group on ring A or B inhibited the enzyme at a low micromolar concentration. Morin with a resorcinol group on ring B as well the flavan-3-ols (+)-catechin and (-)-epicatechin, while bearing a catechol group were much less efficient. Rutin, a 3-O glycosylated quercetin derivative did not inhibit H-DXR. No Triton Flavonoids
With Triton
IC50 (µM)
% of inhibition
% of inhibition
Luteolin
3.4
-
37 a
Quercetin
1.9
-
31 a
-
7b
Rutin Morin
b
ND ND
-
56
Myricetin #
1.5
-
27 a
Baicalein
2.2
-
<5a
Fisetin
3.5
-
21 a
(+)-Catechin
-
40 b
ND
(-)-Epicatechin
-
52 b
ND
Catechol Fosmidomycin*
-
7
0.042
-
b
ND -
Reactions were conducted as described in Experimental. When feasible, IC50 were determined, otherwise the percentages of inhibition at an inhibitor concentration of a 20 µM or b 80 µM are given. (ND not determined). * The IC50 of fosmidomycin was determined as described in [31]. # Illustration of influence of Triton X100 on the inhibition of H-DXR with myricetin (Figure S1).
4
Table 1: Influence of the presence of 0.01% Triton X100 on the inhibition of H-DXR with flavonoids and related compounds
The presence of detergent led to a significant decrease of the inhibition potency (Table 1). It was obvious with baicalein where the inhibition was almost non-existent. When the concentration of flavonoids in the reaction medium varied, no clear dose-dependent inhibition was observed. For example with quercetin, the inhibition remained constant at a level of about 20-25% between 1 to 20 µM flavonoid concentrations. This observation might indicate that a specific inhibition, to some extent, by several flavonoids could not be excluded. As the compounds are susceptible to target the coenzyme binding site, we determined the influence of NADPH on the inhibition level (Table 2). Even at a saturating concentration of NADPH, the protection was only partial, suggesting that flavonoids probably do not bind to the NADPH binding site. Flavonoids
concentration (µM)
% inhibition
% inhibition
No NADPH
160 µM NADPH
Luteolin
6
67
38
Quercetin
4
77
44
Myricetin
4
82
47
Baicalein
4
75
46
Fisetin
6
67
41
The enzyme was pre-incubated during 2 min at 37°C with the inhibitors in the presence or in the absence of NADPH (160 µM final concentration). The enzymatic reaction was initiated by addition of DXP (0.5 mM final concentration) followed by that of NADPH in the first case and by the addition of DXP in the second case.
Table 2: Protection of H-DXR against flavonoid inhibition by NADPH
As the flavonoids did not seem to fit in the cofactor binding site, we studied the influence of the concentration of DXP on the inhibition of the enzyme by myricetin. Our kinetic studies revealed that myricetin behaves as a pure uncompetitive inhibitor of H-DXR versus DXP (Figure 2). The replots (apparent 1/Vmax and 1/Km vs myricetin concentration, see Figure S2) were linear allowing the determination of a Ki value of 1.1 µM [33]. DXR is described to proceed via a sequential ordered Bi-Bi mechanism with NADPH binding first to the enzyme 5
[32]. The uncompetitive character of the inhibition seemed to indicate that DXP is able to bind to the enzyme in the absence of NADPH. This apparent discrepancy implies that, in the presence of NADPH and DXP, the cofactor will bind faster to the enzyme than DXP. 100
1/V0 (min mM-1)
90 80 70 60 50 40 30 20 10 0 -5
0
5
1/[DXP]
10
15
(mM-1)
Reactions were conducted as described in Material and Methods. The enzyme was pre-incubated with DXP (concentration between 0.096 and 0.48 mM) and myricetin at a concentration of 0 (), 0.5 (), 1 () and 2 () µM. The reaction was initiated with NADPH at a final concentration of 160 µM.
Figure 2: Inhibition of H-DXR by myricetin.
3. Discussion Flavonoids were described to inhibit numerous unrelated enzymes. No general rules could be provided for the binding of these molecules to the enzymes except the predominance of hydrophobic interactions between the flavonoids and the enzymes [5]. We tested the influence of some flavonoids on the enzymatic activity of H-DXR with aim to find new inhibitors. We compared a variety of flavonoids possessing similar core structures but differing in single structural elements, such as the presence or absence of a double bond between C-2 and C-3 and the hydroxylation pattern. As the hydroxyl groups may generate hydrogen bonds with amino acids or peptide bonds, the presence or the absence of a hydroxyl group on the carbon skeleton can modulate the inhibitory effect of flavonoids. Half of the tested flavonoids inhibited the H-DXR in a dose-dependent manner in the low micromolar range. The possibility of a non-specific inhibition due to colloidal aggregation of flavonoids is often neglected and a validation by additional proofs than IC50 values to identify them as specific 6
inhibitors is required. In some cases, the specific binding was validated by resolving the crystal structure of the flavonoid-enzyme complex, providing an insight into the binding mode (see Table S2). Other methods, easier to carry out, based on kinetic properties were also proposed as, for instance, the comparison of the inhibition in the presence or the absence of a non-ionic detergent in the reaction medium. It was assumed that the detergent would disperse the aggregates inducing a dramatic decrease of the inhibition in the case of a promiscuous inhibition. In contrast, a weak increase of the IC50 would mean that the inhibition is probably specific. As Triton X100, at least until a concentration of 0.1%, had no effect on the activity of H-DXR, we could compare the inhibition induced by flavonoids in presence and absence of the detergent as proposed earlier [29,30]. A noticeable decrease of inhibition was effectively observed in presence of the detergent. Thus, according to the criteria established by Shoichet and co-workers, the tested flavonoids inhibit probably the enzyme via a non-specific mechanism. The method itself seems not questionable as it was already successfully used to prove the specificity of inhibition of some enzymes [34-39]. The phenomenon was especially noticeable for baicalein since no inhibition was observed in the presence of Triton X100. We could exclude that the absence of inhibition was due to the sequestering of the flavonoid by Triton X100 molecules, as in the case of the human 12-lipoxygenase, baicalein inhibited even in the presence of the detergent [34]. Two other features support a non-specific inhibition of H-DXR by flavonoids. The fact that a pre-incubation of the enzyme with the flavonoids led to an increase of the inhibition, is a necessary though insufficient condition. The lack of protection of NADPH and the uncompetitive inhibition versus DXP are also in favour of an aggregation-based inhibition. Indeed a specific inhibitor should rather be competitive versus the substrate(s). Morin and the flavan-3-ols were ineffective to inhibit H-DXR. In a first approach, these results could be explained for morin, by the presence of a resorcinol instead a catechol group on ring B but also by the failure to give aggregates at the tested concentration [22]. Concerning the tested flavan-3-ols, the loss of the nearly perfect coplanarity existing between ring B and the benzo-γ-pyrone moiety due to the lack of the 2,3 double bond could be responsible for the binding affinity diminution but, as for morin, no formation of aggregates was observed in solution [22]. Moreover, the fact that the tested flavonoids did not inhibit the growth of E. coli (results not shown) strengthens the postulated non-specific inhibition of those compounds.
4. Conclusion 7
The possibility of inhibition by aggregates is often neglected in particular if hydrophobic molecules with very low water solubility are tested. Our results showed that the flavonoids inhibiting the H-DXR can be considered, with high probability, as promiscuous inhibitors. The recently introduced notion of Pan Assay Interference Compounds or PAINS has to be considered in high throughput screening to avoid false positive inhibitors [40]. While described as aggregators, flavonoids, owing to their valuable binding properties, are not to be neglected but particular conditions of assay have to be investigated to be sure that these compounds could be considered as new hits. The addition of detergent in the assays, for instance, would be helpful to prevent false positives in high throughput screening [41,42] and especially when the IC50, in the absence of detergent, are in the micromolar range [43]. It is obvious that affinities in the nanomolar range and high selectivity could restrain the problem of aggregation-based inhibition.
5. Experimental 5.1. Material NADPH was purchased from Sigma-Aldrich. Flavonoids (purity ≥ 95%) were purchased from Sigma-Aldrich, Carl Roth GmbH (Karlsruhe, Germany) or Extrasynthese (Genay, France). They were used without further purification. DXP was synthesized according to Meyer et al.[44]. The cloning and purification of His-tagged DXR (H-DXR) was previously described [31].
5.2. H-DXR activity [31] The assays were performed at 37°C in a 50 mM Tris/HCl buffer pH 7.5 containing 3 mM MgCl2 and 2 mM DTT. The volume was 500 µL. The enzyme was pre-incubated during 2 min with DXP. The enzymatic reaction was initiated by addition of NADPH. The concentrations of DXP and NADPH were 480 µM and 160 µM respectively. The decrease of absorbance at 340 nm due to NADPH oxidation was monitored with an Uvikon 933 UV-Vis spectrophotometer to determine the initial rates. The retained values were the average of at least two measurements. The relative average deviation must be lower than 4 %. The protein concentration was determined by the Bradford method using the Bio-Rad protein assay and bovine serum albumin as the standard [45].
8
5.3. Inhibition studies The DXR inhibition by the flavonoids was quantified by determining the IC50 values. They were obtained by plotting the percentage of residual activity versus the log of inhibitor concentration. In some cases, due the limited solubility of the compounds and/or the low inhibition potential, only the percentage of inhibition at given concentrations (20 or 80 µM) was given. H-DXR was pre-incubated during 2 min in the presence of the inhibitors at different concentrations and DXP (480 µM). NADPH (160 µM final concentration) was then added to measure the residual activity. The flavonoids are poorly soluble in water. Flavonoids solutions at different concentrations were prepared in DMSO by serial dilutions from 100 mM stock solutions. Aliquots of the solutions were added in the assays so that the concentration of DMSO did not exceed 0.4% (v/v). Triton X100 (0.01%) was included in the reaction medium to determine its influence on the inhibition capacity of the tested flavonoids. The inhibition mechanism of myricetin was studied by pre-incubating H-DXR with DXP at varied concentrations (96 to 480 µM) and fixed concentrations of myricetin (0.5, 1 and 2 µM). The enzymatic reaction was initiated by NADPH (final concentration 160 µM). To test the influence of NADPH on the inhibition, the enzyme was pre-incubated with the inhibitors and NADPH (160 µM) during 2 min. DXP (480 µM final concentration) was added to initiate the reaction and determine the residual activity. The H-DXR inhibitor fosmidomycin was used as a positive reference. It was tested as described in [31]. An IC50 value of 42 nM was measured. As the value was near that obtained in earlier studies, we considered that the enzyme was functionally equivalent to that used previously.
5.4. Bacterial growth inhibition The antimicrobial activity of each inhibitor of H-DXR was tested by the paper disc diffusion method on Escherichia coli XL1 Blue [31]. Paper discs were impregnated with 400 nanomoles of inhibitors. Fosmidomycin (10 nanomoles) was the positive reference.
Conflicts of interest The authors declare no conflicts of interest.
Acknowledgements 9
C. Zinglé was a recipient of a grant from the ‘Ministère de la Recherche’. This work was supported by a grant from the “Agence Nationale de la Recherche” (Grant Nb ANR-06BLAN-0291-01).
Supplementary material Table S1: List of enzyme inhibited by flavonoids Table S2: List of X-ray structures of (enzyme, flavonoids) complexes Figure S1: Influence of Triton X100 on the inhibition of H-DXR with myricetin Figure S2: Replot of 1/Km apparent vs myricetin concentration and replot of 1/Vmax apparent vs myricetin concentration
References
[1]
B.H. Havsteen, The biochemistry and medical significance of the flavonoids, Pharmacol. Ther. 96 (2002) 67-202.
[2]
T.P.T. Cushnie, A.J. Lamb, Antimicrobial activity of flavonoids, Int. J. Antimicrob. Agents 26 (2005) 343-356.
[3]
T.P.T. Cushnie, A.J. Lamb, Recent advances in understanding the antibacterial properties of flavonoids, Int. J. Antimicrob. Agents 38 (2011) 99-107.
[4]
National Center for Biotechnology Information - PubChem BioAssay database http://wwwncbinlmnihgov/
[5]
O. Dangles , C. Dufour, Flavonoid-protein interactions in Flavonoids: Chemistry, Biochemistry and Applications Edited by Øyvind M Andersen and Kenneth R Markham, CRC Press (2005) pp 443-469
[6]
M. Rohmer, The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants, Nat. Prod. Rep. 16 (1999) 565-574.
[7]
H. Jomaa, J. Wiesner, S. Sanderbrand, B. Altincicek, C. Weidemeyer, M. Hintz, I. Türbachova, M. Eberl, J. Zeidler, H.K. Lichtenthaler, D. Soldati, E. Beck, Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs, Science 285 (1999) 1573-1576.
[8]
M. Rohmer, C. Grosdemange-Billiard, M. Seemann, D. Tritsch, Isoprenoid biosynthesis as a novel target for antibacterial and antiparasitic drugs, Curr. Opin. Invest. Drugs 5 (2004) 154-162.
10
[9]
M. Okuhara, Y. Kuroda, T. Goto, M. Okamoto, H. Terano, M. Kohsaka, H. Aoki, H. Imanaka, Studies on new phosphonic acid antibiotics. III. Isolation and characterization of FR-31564, FR-32863 and FR-33289, J. Antibiot. 33 (1980) 24-28.
[10]
Y. Shigi, Inhibition of bacterial isoprenoid synthesis by fosmidomycin, a phosphonic acid-containing antibiotic, J. Antimicrob. Chemother. 24 (1989) 131-145.
[11]
T. Kuzuyama, T. Shimizu, S. Takahashi, H. Seto, Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis, Tetrahedron Lett. 39 (1998) 7913-7916.
[12]
A.S. Messiaen, T. Verbrugghen, C. Declerck, R. Ortmann, M. Schlitzer, H. Nelis, S. Van Calenbergh, T. Coenye, Resistance of the Burkholderia cepacia complex to fosmidomycin and fosmidomycin derivatives, Int. J. Antimicrob. Agents 38 (2011) 261-264.
[13]
A. Hemmerlin, D. Tritsch, P. Hammann, M. Rohmer, T.J. Bach, Profiling of defense responses in Escherichia coli treated with fosmidomycin, Biochimie 99 (2014) 54-62.
[14]
M. Andaloussi, L.M. Henriksson, A. Wieckowska, M. Lindh, C. Björkelid, A.M. Larsson, S. Suresh, H. Iyer, B.R. Srinivasa, T. Bergfors, T. Unge, S.L. Mowbray, M. Larhed, T.A. Jones, A. Karlén, Design, synthesis, and X-ray crystallographic studies of α-aryl substituted fosmidomycin analogues as inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, J. Med. Chem. 54 (2011) 4964-4976.
[15]
C.T. Behrendt, A. Kunfermann, V. Illarionova, A. Matheeussen, M.K. Pein, T. Gräwert, J. Kaiser, A. Bacher, W. Eisenreich, B. Illarionov, M. Fischer, L. Maes, M. Groll, T. Kurz, Reverse fosmidomycin derivatives against the antimalarial drug target ispC (Dxr), J. Med. Chem. 54 (2011) 6796-6802.
[16]
C. Zinglé, L. Kuntz, D. Tritsch, C. Grosdemange-Billiard, M. Rohmer, Isoprenoid biosynthesis via the methylerythritol phosphate pathway: structural variations around phosphonate anchor and spacer of fosmidomycin, a potent inhibitor of deoxyxylulose phosphate reductoisomerase, J. Org. Chem. 75 (2010) 3203-3207.
[17]
T. Kurz, D. Geffken, C. Wackendorff, Hydroxyurea analogues of fosmidomycin, Z. Naturforsch 58b (2003) 106-110.
[18]
M. Courtois, Z. Mincheva, F. Andreu, M. Rideau, M.C. Viaud-Massuard, Synthesis and biological evaluation with plant cells of new fosmidomycin analogues containing a benzoxazolone or oxazolopyridinone ring, J. Enzyme Inhib. Med. Chem. 19 (2004) 559-565.
11
[19]
C. Zinglé, L. Kuntz, D. Tritsch, C. Grosdemange-Billiard, M. Rohmer, Modifications around the hydroxamic acid chelating group of fosmidomycin, an inhibitor of the metalloenzyme 1-deoxyxylulose 5phosphate reductoisomerase (DXR), Bioorg. Med. Chem. Lett. 22 (2012) 6563-6567.
[20]
V.T. Athavale, L.H. Prabhu, D.G. Vartak, Solution stability constants of some metal complexes of derivatives of catechol, J. Inorg. Nucl. Chem. 28 (1966) 1237-1249.
[21]
L. Deng, K. Endo, M. Kato, G. Cheng, S. Yajima, Y. Song, Structures of 1deoxy-D-xylulose 5-phosphate reductoisomerase/lipophilic phosphonate complexes, ACS Med. Chem. Lett. 2 (2011) 165-170.
[22]
L. Pohjala, P. Tammela, Aggregating behavior of phenolic compounds-a source of false bioassay results, Molecules 17 (2012) 10774-10790.
[23]
S.L. McGovern, E. Caselli, N. Grigorieff, B.K. Shoichet, A common mechanism underlying promiscuous inhibitors from virtual and highthroughput screening, J. Med. Chem. 45 (2002) 1712-1722.
[24]
S.L. McGovern, B.K. Shoichet, Kinase inhibitors: not just for kinases anymore, J. Med. Chem. 46 (2003) 1478-1483.
[25]
S.L. McGovern, B.T. Helfand, B. Feng, B.K. Shoichet, A specific mechanism of nonspecific inhibition, J. Med. Chem. 46 (2003) 4265-4272.
[26]
J. Seidler, S.L. McGovern, T.N. Doman, B.K. Shoichet, Identification and prediction of promiscuous aggregating inhibitors among known drugs, J. Med. Chem. 46 (2003) 4477-4486.
[27]
K.E.D. Coan, D.A. Maltby, A.L. Burlingame, B.K. Shoichet, Promiscuous aggregate-based inhibitors promote enzyme unfolding, J. Med. Chem. 52 (2009) 2067-2075.
[28]
A. Whitty, Growing PAINS in academic drug discovery, Future Med. Chem. 3 (2011) 797-801.
[29]
B.Y. Feng, B.K. Shoichet, A detergent-based assay for the detection of promiscuous inhibitors, Nat. Protoc. 1 (2006) 550-553.
[30]
A.J. Ryan, N.M. Gray, P.N. Lowe, C. Chung, Effect of detergent on “promiscuous” inhibitors, J. Med. Chem. 46 (2003) 3448-3451.
[31]
L. Kuntz, D. Tritsch, C. Grosdemange-Billiard, A. Hemmerlin, A. Willem, T.J. Bach, M. Rohmer, Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase, Biochem. J. 386 (2005) 127135.
12
[32]
A.T. Koppisch, D.T. Fox, B.S. Blagg, C.D. Poulter, E. coli MEP synthase: steady-state kinetic analysis and substrate binding, Biochemistry 41 (2002) 236-243.
[33]
I.H. Segel, Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems, Wiley-Interscience, NewYork; 1975: 161226.
[34]
J.D. Deschamps, V.A. Kenyon, T.R. Holman, Baicalein is a potent in vitro inhibitor against both reticulocyte 15-human and platelet 12-human lipoxygenases, Bioorg. Med. Chem. 14 (2006) 4295-4301.
[35]
R.F. Freitas, I.M. Prokopczyk, A. Zottis, G. Oliva, A.D. Andricopulo, M.T.S. Trevisan, W. Vilegas, M.G.V. Silva, C.A. Montanari, Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors, Bioorg. Med. Chem. 17 (2009) 2476-2482.
[36]
M. Brunskole, K. Zorko, V. Kerbler, S. Martens, J. Stojan, S. Gobec, T. Lanisnik Rizner, Trihydroxynaphthalene reductase of Curvularia lunata - a target for flavonoid action?, Chem. Biol. Interact. 178 (2009) 259-267.
[37]
S. Arioka, M. Sakagami, R. Uematsu, H. Yamaguchi, H. Togame, H. Takemoto, H. Hinou, S.-I. Nishimura, Potent inhibitor scaffold against Trypanosoma cruzi trans-sialidase, Bioorg. Med. Chem. 18 (2010) 16331640.
[38]
T. Tarragó, N. Kichik, B. Claasen, R. Prades, M. Teixidó, E. Giralt, Baicalein, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor, Bioorg. Med. Chem. 16 (2008) 7516-7524.
[39]
D. Duan, A. K. Doak, L. Nedyalkova, B. K. Shoichet, Colloidal aggregation and the in vitro activity of traditional chinese medicines, ACS Chem. Biol (2015) in press
[40]
J.B. Baell, G.A. Holloway, New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays, J. Med. Chem. 53 (2010) 2719-2740.
[41]
A. Fallarero, L. Hanski, P. Vuorela, How to translate a bioassay into a screening assay for natural products: General considerations and implementation of antimicrobial screens, Planta Med. 80 (2014) 1182-1199.
[42]
X. Xie, L. Sun, Z.Y. Pesetto, Y. Zhao, Z. Zang, L. Zhong, M. Wu, Q. Su, X. Gao, W. Zan, Y. Sun, Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators, PLoS One 8 (2013) e78042.
13
[43]
C. Zinglé, D. Tritsch, C. Grosdemange-Billiard, M. Rohmer, Catecholrhodanine derivatives: Specific and promiscuous inhibitors of Escherichia coli deoxyxylulose phosphate reductoisomerase (DXR), Bioorg. Med. Chem. 22 (2014) 3713-3719.
[44]
O. Meyer, J.-F. Hoeffler, C. Grosdemange-Billiard, M. Rohmer Practical synthesis of 1-deoxy-D-xylulose and 1-deoxy-D-xylulose 5-phosphate allowing deuterium labelling, Tetrahedron 60 (2004) 12153-12262.
[45]
M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle protein-dye binding, Anal. Biochem. 72 (1976) 248-254.
14
Tables Table 1
Influence of the presence of 0.01% Triton X100 on the inhibition of H-DXR with flavonoids and related compounds
No Triton Flavonoids
With Triton
IC50 (µM)
% of inhibition
% of inhibition
Luteolin
3.4
-
37 a
Quercetin
1.9
-
31 a
-
7b
Rutin Morin
b
ND
-
56
Myricetin
1.5
-
27 a
Baicalein
2.2
-
<5a
Fisetin
3.5
-
21 a
(+)-Catechin
-
40 b
ND
(-)-Epicatechin
-
52 b
ND
Catechol
-
7b
ND
0.042
-
-
Fosmidomycin*
ND
Reactions were conducted as described in Experimental. When feasible, IC50 were determined, otherwise the percentages of inhibition at an inhibitor concentration of a 20 µM or b 80 µM are given. (ND not determined). * The IC50 of fosmidomycin was determined as described in [31].
15
Table 2
Protection of H-DXR by NADPH against flavonoid inhibition
Flavonoids
concentration (µM)
% inhibition
% inhibition
No NADPH
160 µM NADPH
Luteolin
6
67
38
Quercetin
4
77
44
Myricetin
4
82
47
Baicalein
4
75
46
Fisetin
6
67
41
The enzyme was pre-incubated during 2 min at 37°C with the inhibitors in the presence or in the absence of NADPH (160 µM final concentration). The enzymatic reaction was initiated by addition of DXP (0.5 mM final concentration) followed by that of NADPH in the first case and by the addition of DXP in the second case.
16
Figures
Figure 1: Flavonoids tested on His-tagged DXR of E. coli
17
100
1/V0 (min mM-1)
90 80 70 60 50 40 30 20 10 0 -5
0
5
10
15
1/[DXP] (mM-1)
Figure 2: Inhibition of H-DXR by myricetin. Reactions were conducted as described in Material and Methods. The enzyme was preincubated with DXP (concentration between 0.096 and 0.48 mM) and myricetin at a concentration of 0 (), 0.5 (), 1 () and 2 () µM. The reaction was initiated with NADPH at a final concentration of 160 µM.
18
Graphical abstract
19
Highlights Possible erroneous conclusions regarding the inhibition of enzymes by flavonoids Hydrophobic molecules can generate aggregates responsible of enzyme inhibition Inhibition studies with flavonoids require additional testing with non-ionic detergent Flavonoids inhibit deoxyxylulose phosphate reductoisomerase via a non-specific way
20