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Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids
Accepted Manuscript Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids Daniela Vullo, Sonia Del Prete, Clemente Capasso, Claudiu T. Supuran PII: DOI: Reference:
S0960-894X(16)30078-6 http://dx.doi.org/10.1016/j.bmcl.2016.01.078 BMCL 23543
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
4 January 2016 25 January 2016 28 January 2016
Please cite this article as: Vullo, D., Prete, S.D., Capasso, C., Supuran, C.T., Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.078
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Carbonic anhydrase activators: Activation of the -carbonic anhydrase from Malassezia globosa with amines and amino acids Daniela Vullo,a Sonia Del Prete, b,c Clemente Capasso,b* and Claudiu T. Supurana,c* a
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della
Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy. b
Istituto di Biochimica delle Proteine – CNR, Via P. Castellino 111, 80131 Napoli, Italy.
c
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm.
188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. The -carbonic anhydrase (CA, EC 4.2.1.1) from the dandruff producing fungus Malassezia globosa, MgCA, was investigated for its activation with amines and amino acids. MgCA was weakly activated by amino acids such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3 M) but more effectively activated by D-Phe, LDOPA, L-Trp, histamine, dopamine, pyridyl-alkylamines, and 4-(2-aminoethyl)-morpholine), with KAs of 5.82-10.9 M. The best activators were L-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81 M. This study may help a better understanding of the activation mechanisms of -CAs from pathogenic fungi as well as the design of tighter binding ligands for this enzyme which is a drug target for novel types of anti-dandruff agents.
Keywords: carbonic anhydrase; activator; amine; amino acid; dandruff; Malassezia globosa
______ * Corresponding authors. Tel/Fax: +39-0816132559. E-mail: [email protected] (Clemente Capasso); Tel/Fax: +39-055-4573729, E-mail:[email protected] (Claudiu T. Supuran).
1
Various species of the fungal pathogen Malassezia are the ethiologic agents of dandruff, a condition affecting billions of persons.1 Dandruff is triggered by several factors, including an increase in sebum production, irritation by pathogenic organisms (particularly fungi belonging to the above mentioned genus), individual susceptibility factors and stress. 1 The recent characterization of a carbonic anhydrase (CA, EC 4.2.1.1) belonging to the -class in Malassezia globosa.
1,2
MgCA, led us to initiate a systematic search for modulators of this enzyme activity, i.e., inhibitors and activators, which might show therapeutic potential. 2,3 Of the six independently-evolved (, , , , and η) CA classes discovered to date, only and -CAs were reported so far in fungi:2-5 basidiomycetes and hemiascomycetouos yeasts contain only -CAs, whereas the filamentous ascomycetes also possess -CAs in addition to the -class enzymes.2a Yet, the CAs play crucial functions in the life cycles of pathogenic fungi, among which CO2 sensing, by coupling with an adenylyl cyclase, which allows the pathogen to adapt to the dramatic changes in CO2 levels during colonization and infection of the human host and to fine-tune its virulence in the various physiologic conditions in which it grows. 3-5 Muhlschlegel’s group showed3a that inhibition with ethoxzolamide, a strong sulfonamide CA inhibitor, of the -CAs from fungal pathogenic species such as Candida albicans or Cryptococcus neoformans lead, in certain conditions, to the inhibition of the growth of the pathogen, whereas our group showed that inhibition of MgCA with various sulfonamide inhibitors has the same effect,2b leading to an impairment of the pathogen growth with fragmented fungal hyphae, a situation also observed after the treatment with ketoconazole, a clinically used antifungal drug acting through a different mechanism of action.2b CAs are present in many pathogenic organisms apart fungi, such as bacteria and protozoa, where they play crucial roles in the microbial virulence. 6,7 The CA distribution pattern in bacteria is very intriguing,as some bacteria encode CAs belonging just to one class, others from two and even three different genetic classes (i.e., α-, β- and γ-CAs), whereas the protozoa encode for α-, β- and ηclass enzymes, some of which were recently shown to be drug targets.7,8 Inhibition of CAs, mainly of the mammalian -class enzymes, was investigated in great detail with several clinically used CA inhibitors (CAIs) having applications as diuretics, antiglaucoma, antiobesity or anticancer agents/diagnostic tools. 6 The inhibition of fungal CAs, such as the Candia albicans, Candida glabrata, Cryptococcus neoformans and Saccharomyces cerevisiae enzymes with sulfonamides, carboxylates and dithiocarbamates was also investigated in the lat period,3,9,10 in the search of antifungals with a different mechanism of action, considering the serious drug resistance problems with the presently used clinical agents belonging to most antifungal classes.2,3,10 2
On the other hand, CA activators (CAAs), were less investigated in pathogenic organisms, as this class of enzyme modulators started to be investigated systematically for their interaction with mammalian -CAs only in the past years.11-14 Indeed, our group reported kinetic and X-ray crystallographic studies for the interaction of all human CA isoforms (hCA I – XV) with amino acid and amine activators, also unravelling the activation mechanism with this class of modulators of activity.11a-e,12-14 Although the mammalian enzymes CAAs do not have clinical applications for the moment, recent work from Muller’s group11f,g highlighted the important role that the CA activators play in modulating the calcification of the bone, opening interesting potential application for the modulators of CA activity for obtaining artificial tissues. Recently we have extended the CA activation studies to the -class enzymes from pathogenic fungi, reporting the first such studies for S. cerevisiae, ScCA15a (encoded by the NCE103 gene found in all basidiomycetes and hemiascomycetous yeasts),2 C. glabrata, CgCA,15b and C. albicans CaCA enzymes.15c These studies may bring interesting information regarding the physiologic roles of these enzymes, which seem to be involved in metabolic, biosynthetic processes (e.g. fatty acid biosynthesis) 16 apart the chemosensing roles mentioned above.4,5 All CA classes have a metal hydroxide cofactor within the active center, which is crucial for catalysis.2,3,6,7 The CA activation mechanism considers the rate-determining step of the CA catalytic cycle (for the hydration of CO2 to bicarbonate), which is the formation of the zinc hydroxide species of the enzyme (zinc is the prevalent metal ion in various CA classes, being the natural one in the fungal CAs).6a,c,11 This process occurs via the transfer of a proton from the Zn(II)-coordinated water molecule to the environment and is assisted by amino acid residues from the enzyme active site or by the binding of an activator molecule within the enzyme cavity.11-15 For the -CAs these phenomena are well understood, as many X-ray crystal structures of enzyme-activator adducts are available.11,12,14 Although few studies that have been published to date for other CA classes, there is evidence that activation mechanisms of the - and -CAs are similar.15 For example, the binding of the CA activator within the enzyme active site, which leads to the formation of an enzyme – activator complex seems to occur also in - and -CAs investigated to date.11-15 This adduct facilitates the shuttling of protons between the Zn(II) ion-coordinated water molecule and the environment, generating thus the nucleophilic zinc hydroxide, which is the catalytically active species of the enzymes.11-15 Here we report the first activation study of MgCA with a series of amines and amino acids (of types 1-19, Fig. 1), which were investigated earlier
11-14
for their interactions with mammalian
-CAs as well as more recently with the - and -class enzymes from the Archaea domain and from pathogenic fungi, respectively.15 This study may help a better understanding of the -CA 3
catalytic/activation mechanism, as the natural proton shuttling residue in this class of enzymes has not been yet identified, unlike the α-CAs for which it is known that a His residue placed in the middle of the active site cavity (His64, hCA I numbering) plays this function.6 O
O
O
H2N
H2N
OH
H2N
OH
OH OH
N N 1: L-His 2: D-His
3: L-Phe 4: D-Phe
5: L-DOPA 6: D-DOPA
O
O
O
H2N
H2N
OH
H2N
OH
OH
OH
N NH2
OH 9: L-Tyr 10: D-Tyr
7: L-Trp 8: D-Trp
N
11: 4-H2N-L-Phe
NH2
NH2
NH2
HO
N H
N H
OH
12 OH
14
13 H N
NH2 ( )n
15: n = 1 16: n = 2
HO X
OH H N
N NH2 17: X = NH 18: X = O
HO 19
Fig. 1: Structure of activators 1-19 investigated in the present study. Data of Table 117 show histamine to be an activator of MgCA as well as hCA II and β-class enzymes from other fungal pathogens, investigated earlier.15 At a concentration of 10 M, histamine enhanced kcat values for all the enzymes, whereas the KM values remain unchanged. According to previous studies, histamine is a millimolar activator for the -class enzyme (hCA II), with KA of 125 M,11 whereas it is a more effective activator for the fungal ones, such as the yeast ScCA (KA of 20.4 M) or C. glabrata CgCA enzymes (KA of 27.4 M).15 The results from the present study show that histamine has a higher affinity for MgCA with a KA of 10.9 ± 0.9 M (see discussion later in the text) compared to the other yeast/fungal enzymes. It is thus obvious that the activation mechanisms of the - and -CAs are likely to be similar, i.e., the CA activator enhances
4
kcat but has no influence on KM, facilitating the release of the proton from the water coordinated to the catalytic zinc ion. Table 1: Kinetic parameters for the activation of human hCA II (-class) and -class enzymes from S. cerevisiae (scCA),15a C. glabrata (CgCA),15b and M. globosa (MgCA) with histamine (Hst) 12, measured at 25 °C, pH 8.3 in 20 mM Tris buffer and 20 mM NaClO 4, for the CO2 hydration reaction, MgCA concentration in the assay system was of 12.1 nM. 16
Isozyme
kcat*
KM*
(kcat)Hst**
KA*** (M)
(s-1)
(mM)
(s-1)
Hst
hCA IIa
1.4x106
9.3
2.0x106
125
ScCAb
9.4 x105
9.5
19.6 x105
20.4
CgCAb
3.8 x105
7.9
10.7 x105
c
MgCA
(9.2±0.1)x10
5
11.1±0.1
(18.5 ±0.3) x10
27.4 5
10.9 ±0.9
_______________________________________________________________________________ * Observed catalytic rate without activator. KM values in the presence and the absence of activators were the same for the various CAs (data not shown). ** Observed catalytic rate in the presence of 10 M activator. *** The activation constant (KA) for each enzyme was obtained by fitting the observed catalytic enhancements as a function of the activator concentration. 16 Mean from at least three determinations by a stopped-flow, CO2 hydrase method.16 Standard errors were in the range of 5-10 % of the reported values. a
Human recombinant enzyme, data from ref. 14
b
Yeast/fungal recombinant enzymes data from ref. 15a,b
c
This work. Mean ± standard error (from 3 different assays).
Data of Table 2 show that all amino acids and amines 1-19, which have been investigated in this study, act as CAAs against the fungal enzyme MgCA. However, the activation profiles of these compounds against MgCA are different from other recently investigated - and -class enzymes, such as hCA II, ScCA and CgCA,11,15 which were included in Table 2 for comparison. The following structure activity relationship (SAR) can be observed for the activation of MgCA with compounds 1-19:17-20 (i) Several amino acids, such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr, 4-amino-Lphenylalanine, as well as serotonin 14, showed activation constants in the range of 12.5 – 29.3 M, therefore they are considered as moderate MgCA activators. However, it may be observed that most of these derivatives (except L-Phe, L-/D-Tyr and D-Trp) are much weaker activators of the C.
5
glabrata enzyme CgCA. ScCA on the other hand has completely different activation profiles with these derivatives,being poorly activated by all amino acids (but slightly more susceptible to activation by serotonin). hCA II is highly activated by most amino acids, sometimes with activation constants in the nanomolar range 11-14 (e.g., L-/D-Phe; L-/D-Tyr), see Table 2. Table 2: Activation constants of hCA II (cytosolic α-isozyme), yeast -CAs from S. cerevisiae (ScCA), C. glabrata (CgCA) and MgCA (Malassezia globosa) with amino acids and amines 1 – 19. Activation data of hCA II, ScCA and CgCA with these compounds are retrieved from refs.14,15a,b No.
Compound
KA (M)* hCA IIa
ScCAb
CgCAc
MgCAd
1
L-His
10.9
82
37.0
29.3 ± 1.2
2
D-His
43
85
21.2
18.1 ± 0.9
3
L-Phe
0.013
86
24.1
34.1 ± 2.7
4
D-Phe
0.035
86
15.7
10.7 ± 0.8
5
L-DOPA
11.4
90
23.3
8.31 ± 0.6
6
D-DOPA
7.8
89
15.1
13.7 ± 1.1
7
L-Trp
27
91
22.8
10.1 ± 0.6
8
D-Trp
12
90
12.1
12.5 ± 1.2
9
L-Tyr
0.011
85
9.5
15.7 ± 1.0
10
D-Tyr
0.058
84
7.1
25.1 ± 1.9
11
4-H2N-L-Phe
0.15
21.3
31.6
13.4 ± 0.8
12
Histamine
125
20.4
27.4
10.9 ± 0.9
13
Dopamine
9.2
13.1
27.6
9.43± 0.7
14
Serotonin
50
15.0
16.7
14.2 ± 1.3
15
2-Pyridyl-methylamine
34
16.2
15.0
6.12 ± 0.3
16
2-(2-Aminoethyl)pyridine 15
11.2
16.3
7.30 ± 0.3
17
1-(2-Aminoethyl)-piperazine
9.3
14.9
0.81 ± 0.07
18
4-(2-Aminoethyl)-morpholine
10.2
10.1
5.82 ± 0.4
19
L-Adrenaline
0.95
10.8
0.72 ± 0.05
2.3 0.19
96
* Mean from three determinations by a stopped-flow, CO2 hydrase method.25 Standard errors were in the range of 5-10 % of the reported values for the activation of hCA II, ScCA and CgCA. 21,23,24 a
Human recombinant isozyme, from ref. 21
b
Recombinant archaeal enzyme, from ref. 23
c
Recombinant yeast enzyme, from ref. 24
d
Recombinant yeast enzyme, this study. Mean ± standard error (from 3 different assays). 6
(ii) A second group of derivatives, including D-Phe, L-DOPA, L-Trp, as well as amines 12, 13, 15, 16 and 18, acted as more effective MgCA activators when compared to the compounds discussed above, with activation constants ranging between 5.82 – 10.9 M. Thus, it may be observed that the amines were generally better MgCA activators compared to the carboxylic acids with which they are structurally related (compare Histamine with L/D-His, or dopamine with L-/DDOPA, for example). The heterocylic amines incorporating pyridyl, piperazine, and morpholine rings were more effective MgCA activators compared to histamine, dopamine or serotonin. (iii) The most potent MgCA activators were L-adrenaline 19 and 1-(2-aminoethyl)piperazine 17, with activation constants of 0.72-0.81 M. Thus, small structural modifications in the scaffold of the amine activator (e.g., compare dopamine 13 with L-adrenaline 19) lead to drastic changes of the activating effect, with 19 being 13-times a better activator compared to 13.
OH
OH
O O
H
N
+ H
H
H O H H N
+
O
NH2
Arg51
O
H2 N
Asp49
H
H O
Cys47
- Zn S
2+
S
Cys106
N N H
His103
Fig. 2: Proposed schematic interactions between an activator (L-DOPA, 5) and the MgCA active site. The Zn(II) ion is coordinated by two Cys and one His residue, and by a water molecule (M. globosa CA numbering of amino acid residues).2b Both the amino and carboxylate moieties of the activator 5 can participate in hydrogen bonds with amino acid residues in the neighborhood of the metal ion center and facilitate the transfer of the proton from the zinc-bound water to the 7
environment with generation of the zinc hydroxide species of the enzyme. A network of hydrogen bonds between the bridging water molecule interacting with the activator and the Asp49 - Arg51 catalytic dyad and zinc coordinated water has been hypothesized here, but the real number of water molecules participating in the activation process is unknown. In hCA II – activator crystal structures it has been shown to be between 1 and 3 water molecules. 11,14 A possible activation mechanism of the M. globosa -CAs by L-DOPA 5 is depicted schematically in Fig. 2. As for other -CAs, the catalytic Zn(II) ion in the MgCA active site is coordinated by two Cys and one His residues (Cys47, His103 and Cys106, MgCA numbering system).2b A second pair of conserved amino acid residues in all sequenced -CAs known to date,2-7 is constituted by the dyad Asp49 – Arg51 (M. globosa CA numbering). These amino acids are close2-4 to the zinc-bound water molecule, which is the fourth Zn(II) ligand in this type of open active site -CAs,2 and participate in a network of hydrogen bonds, which probably assist the water deprotonation and the formation of the nucleophilic, zinc hydroxide species of the enzyme. 8,10 The active site channel of -CAs (as exemplified by the X-ray crystal structure of the C. neoformans enzyme Can23a or Vibrio cholerae
21
enzymes) is a channel which can accommodate elongated
molecules such as the aromatic amino acids/amines which have been investigated here. Thus, we hypothesize that the activator (such as L-DOPA 5) may bind closely to the active site channel, establishing supplementary hydrogen bonds with the polar moieties of amino acid residues such as the catalytic dyad mentioned above, or with the zinc-bound water molecule (directly or through a network of several other water molecules, as demonstrated for the interaction of -CAs with this type of activator)11,14 and thus assist water deprotonation and facilitate the catalytic turnover. Indeed, both the amino or carboxyl moieties of the activators can establish hydrogen bonds with these structural elements, due to the presence of many heteroatoms in their molecules. Fig. 2 shows schematically a putative binding mode of L-DOPA 2 within the active site of MgCA. This hypothesis should be confirmed by X-ray crystallography, however the structure of MgCA has not yet been resolved. In conclusion, we report the first activation study of the -CA from the fungal pathogen M. globosa with amines and amino acids. MgCA was weakly activated by amino acids such as L-/DHis, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3 M) but more effectively activated by D-Phe, L-DOPA, L-Trp, histamine, dopamine, pyridyl-alkylamines, and 4(2-aminoethyl)-morpholine), with KAs of 5.82-10.9 M. The best activators were L-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81 M. This study may help a better understanding of the activation mechanisms of -CAs from pathogenic fungi as well as the 8
design of tighter binding ligands for this enzyme which is a drug target for novel types of antidandruff agents. Acknowledgments. This research was financed in part by a grant of the 7th Framework Programme of the European Union (Dynano project).
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References and Notes
1. a) Clavaud, C.; Jourdain, R.; Bar-Hen, A.; Tichit, M.; Bouchier, C.; Pouradier, F.; El Rawadi, C.; Guillot, J.; Ménard-Szczebara, F.; Breton, L.; Latgé, J.P.; Mouyna, I. PLoS One. 2013, 8, e58203; b) Donnarumma, G.; Perfetto, B.; Paoletti, I.; Oliviero, G.; Clavaud, C.; Del Bufalo, A.; Guéniche, A.; Jourdain, R.; Tufano, M.A.; Breton, L. Arch. Dermatol. Res. 2014, 306, 763. 2. a) Elleuche, S.; Poggeler, S. Curr. Genet. 2009, 55, 211; b) Hewitson, K.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C.T. J. Med. Chem. 2012, 55, 3513; c) Del Prete, S.; De Luca, V.; Vullo, D.; Osman, S.M.; AlOthman, Z.; Carginale, V.; Supuran, C.T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2016, 31, in press (doi: 10.3109/14756366.2015.1102137); d) Del Prete, S.; Vullo, D.; Osman, S.M.; AlOthman, Z.; Capasso, C.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2015, 25, 5194. 3. a) Schlicker, C.; Hall, R.A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C.T.; Muhlschlegel, F.A.; Steegborn, C. J. Mol. Biol. 2009, 385, 1207; b) Hall, R.A.; Mühlschlegel, F.A. Fungal and nematode carbonic anhydrases: Their inhibition in drug design. In Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications, Supuran, C.T.; Winum, J.Y. Eds., John Wiley & Sons, Hoboken, 2009, pp. 301 – 322. 4. a) Klengel, T.; Liang, W.J.; Chaloupka, J.; Ruoff, C.; Schropel, K.; Naglik, J.R.; Eckert, S.E.; Mogensen, E.G.; Haynes, K.; Tuite, M.F.; Levin, L.R.; Buck, J.; Mühlschlegel, F.A. Curr. Biol. 2005, 15, 2021; b) Bahn, Y.S.; Cox, G.M:, Perfect, J.R.; Heitman, J. Curr. Biol. 2005, 15, 2013. 5. a) Mogensen, E.G.; Janbon, G.; Chaloupka, J.; Steegborn, C.; Fu, M.S.; Moyrand, F.; Klengel, T.; Pearson, D.S.; Geeves, M.A., Buck, J.; Levin, L.R.; Mühlschlegel, F.A. Eukaryot. Cell 2006, 5, 103; b) Bahn, Y.S.; Mühlschlegel, F.A. Curr. Opin. Microbiol. 2006, 9, 572. 6. a) Supuran, C.T. Nat. Rev. Drug. Discov. 2008, 7, 168; b) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2016, 31, in press (doi: 10.3109/ 14756366.2015.1122001); c) Supuran, C.T. Expert Opin. Ther. Pat.2013, 23, 677; c) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; d) Supuran, C.T. Curr. Top. Med. Chem. 2007, 7, 825; e) Supuran, C.T.; Scozzafava, A.; Mastrolorenzo, A. Expert Opin. Ther. Pat. 2001, 11, 221. 7. a) Tripp, B.C.; Smith, K.S.; Ferry, J.G. J. Biol. Chem. 2001, 276, 48615; b) Smith, K.S.; Ferry, J.G. FEMS Microbiol. Rev. 2000, 24, 335; c) Zimmerman, S.A.; Ferry, J.G.; Supuran, C.T. Curr. Top. Med. Chem. 2007, 7, 901; d) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; e) Del Prete, S.; Vullo, D.; Fisher, G.M.; Andrews, K.T.; Poulsen, S.A.; Capasso, C.; Supuran, C.T. Bioorg. Med. Chem. Lett.2014, 24, 4389; f) Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.; Scozzafava, A.; Dedhar, S.; Winum, J.Y.; Supuran, C. T. J. Med. Chem. 2011, 54, 8271.
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8. a) Supuran, C. T. Front Pharmacol 2011, 2, 34; b) Capasso, C.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379; c) Capasso, C.; Supuran, C.T. Expert Opin. Ther. Targets 2015, 19, 1689; d) Supuran, C.T.; Capasso, C. Expert Opin. Ther. Targets 2015, 19, 551; d) Capasso, C.; Supuran, C.T. Expert Opin. Ther. Pat. 2013, 23, 693; e) Capasso, C.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2015, 30, 325; f) Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C.T. Curr. Pharm. Des. 2008, 14, 622. 9. a) Aguilera, J.; Van Dijken, J. P.; De Winde, J. H.; Pronk, J. T., Biochem. J. 2005, 391, 311; b) Aguilera, J.; Petit, T.; de Winde, J.; Pronk, J. T. FEMS Yeast Res. 2005, 5, 579. 10. a) Innocenti, A.; Mühlschlegel, F.A.; Hall, R.A.; Steegborn, C.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2008, 18, 5066; b) Innocenti, A.; Hall, R.A.; Schlicker, C.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. 2009, 17, 2654; c) Innocenti, A.; Hall, R.A.; Schlicker, C.; Scozzafava, A.; Steegborn, C.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. 2009, 17, 4503; d) Innocenti, A.; Leewattanapasuk, W.; Mühlschlegel, F.A.; Mastrolorenzo, A.; Supuran, C.T.. Bioorg. Med. Chem. Lett. 2009, 19, 4802; e) De Simone, G.; Di Fiore, A.; Supuran, C.T. Curr. Pharm. Des. 2008, 14, 655. 11. a) Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C.T. Biochemistry, 1997, 36, 10384; b) Temperini, C.; Scozzafava, A.; Supuran, C.T. Curr. Pharm. Des. 2008, 14, 708; c) Ilies, M.; Scozzafava, A.; Supuran, C.T. Carbonic anhydrase activators. In Carbonic anhydrase - Its inhibitors and activators, Supuran, C.T.; Scozzafava, A.; Conway J. Eds., CRC Press, Boca Raton (FL), USA, 2004, pp. 317-352; d) Supuran, C.T.; Scozzafava, A. Activation of carbonic anhydrase isozymes. In The Carbonic Anhydrases - New Horizons, Chegwidden, W.R.; Carter, N.; Edwards, Y. Eds.; Birkhauser Verlag: Basel, Switzerland, 2000, pp. 197-219; e) Supuran, C.T. Therapy, 2007, 4, 355; f) Wang, X.; Schröder, H.C.; Müller, W.E. Int. Rev. Cell. Mol. Biol. 2014, 313, 27; g) Wang, X.; Schröder, H.C.; Schlossmacher, U.; Neufurth, M.; Feng, Q.; Diehl-Seifert, B.; Müller, W.E. Calcif. Tissue Int. 2014, 94, 495. 12. a) Scozzafava, A.; Supuran, C.T. J. Med. Chem. 2002, 45, 284; b) Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2002, 12, 1177; c) Ilies, M.; Banciu, M.D.; Ilies, M.A.; Scozzafava, A.; Caproiu, M.T.; Supuran, C.T. J. Med. Chem. 2002, 45, 504; d) Scozzafava, A.; Iorga, B.; Supuran, C.T. J. Enzyme Inhib. 2000, 15, 139; e) Temperini, C.; Scozzafava, A.; Vullo, D.; Supuran, C.T. J. Med. Chem. 2006, 49, 3019. 13. a) Parkkila, S.; Vullo, D.; Puccetti, L.; Parkkila, A.K.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem Lett. 2006, 16, 3955; b) Vullo, D.; Nishimori, I.; Innocenti, A.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2007, 17, 1336; c) Clare, B.W.; Supuran, C.T. J. Pharm.
11
Sci. 1994, 83, 768; d) Saada, M.C.; Montero, J.L.; Winum, J.Y.; Vullo, D.; Scozzafava, A.; Supuran, C.T. J. Med. Chem. 2011, 54, 1170. 14. a) Temperini, C.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2007, 17, 628; b) Vullo, D.; Nishimori, I.; Innocenti, A.; Scozzafava, A.; C.T. Supuran, Bioorg. Med. Chem. Lett. 2007, 17, 1336; c) Vullo, D.; Innocenti, A.; Nishimori, I.; Scozzafava, A.; Kaila, K.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2007, 17, 4107; d) Pastorekova, S.; Vullo, D.; Nishimori, I.; Scozzafava, A.; Pastorek, J.; Supuran, C.T. Bioorg. Med. Chem. 2008, 16, 3530. 15. a) Isik, S.; Kockar, F.; Aydin, M.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A..; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2009, 19, 1662; b) Innocenti, A.; Leewattanapasuk, W.; Manole, G.; Scozzafava, A.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2010, 20, 1701; c) Innocenti, A.; Hall, R.A.; Scozzafava, A.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. 2010, 18, 1034; d) Innocenti, A.; Zimmerman, S.A.; Scozzafava, A.; Ferry, J.G.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2008, 18, 6194; e) Supuran, C.T.; Clare, B.W. Eur. J. Med. Chem. 1999, 34, 41. 16. a) Kim, M.S.; Ko, Y.J.; Maeng, S.; Floyd, A.; Heitman, J. Bahn,; Y.S. Genetics 2010, 185, 1207; b) Lehneck, R.; Pöggeler, S. Appl. Microbiol. Biotechnol. 2014, 98, 8433; c) Lehneck, R.; Neumann, P.; Vullo, D.; Elleuche, S.; Supuran, C.T.; Ficner, R.; Pöggeler, S. FEBS J.2014, 281, 1759; d) Mastrolorenzo, A.; Rusconi, S.; Scozzafava, A.; Barbaro, G.; Supuran, C.T. Curr. Med. Chem. 2007, 14, 2734; e) Bayram, E.; Senturk, M.; Kufrevioglu, O.I.; Supuran, C.T. Bioorg. Med. Chem. 2008, 16, 9101. 17. Khalifah, R.G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-flow instrument was used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, 10 – 20 mM Hepes (pH 7.5, for α-CAs) or Tris (pH 8.3, for β-CAs) as buffers, and 20 mM Na2SO4 or 20 mM NaClO4 (for maintaining constant the ionic strength), following the CA-catalyzed CO2 hydration reaction for a period of 10 s at 25°C. The CO 2 concentrations ranged from 0.17 to 17 mM for the determination of the kinetic parameters and activation constants. For each activator at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activators 1-19 (10 mM) were prepared in distilled-deionized water and dilutions up to 0.001 M were done thereafter with distilled-deionized water. Activator and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-A complex. The activation constant (KA), defined similarly with the inhibition
12
constant KI,18-20 can be obtained by considering the classical Michaelis-Menten equation (equation 1), which has been fitted by non-linear least squeares by using PRISM 3: v = vmax /1 + KM /[S]x(1 + [A]f/KA)
(1)
where [A] f is the free concentration of activator. Working at substrate concentrations considerably lower than K M ([S] << KM), and considering that [A] f can be represented in the form of the total concentration of the enzyme ([E] t) and activator ([A]t), the obtained competitive steady-state equation for determining the activation constant is given by eq. 2:18-20 v = v0 . KA /KA + ([A]t – 0.5 ([A]t + [E]t + KA) – ([A]t + [E]t + KA)2 – 4[A]t.[E]t)1/2 (2) where v0 represents the initial velocity of the enzyme-catalyzed reaction in the absence of activator.18-20 The concentration of the enzymes in the assay system were in the range of 10-14 nM. 18. a) Temperini, C.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2006, 16, 5152; b) Temperini, C.; Innocenti, A.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. 2008, 16, 8373; c) Vullo, D.; Voipio, J.; Innocenti, A.; Rivera, C.; Ranki, H.; Scozzafava, A.; Kaila, K.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2005, 15, 971; d) Nishimori, I.; Minakuchi, T.; Morimoto, K.; Sano, S.; Onishi, S.; Takeuchi, H.; Vullo, D.; Scozzafava, A.; Supuran, C.T. J. Med. Chem. 2006, 49, 2117. 19. a) Abdo, M.R.; Vullo, D.; Saada, M.C.; Montero, J.L.; Scozzafava, A.; Winum, J.Y.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2009, 19, 2440; b) Saada, M.C.; Montero, J.L.; Vullo, D.; Scozzafava, A.; Winum, J.Y.; Supuran, C.T. Bioorg. Med. Chem. 2014, 22, 4752; c) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C.T. J. Med.Chem. 2010, 53, 5511. 20. a) Dave, K.; Scozzafava, A.; Vullo, D.; Supuran, C.T.; Ilies, M.A. Org. Biomol. Chem. 2011, 9, 2790; b) Draghici, B.; Vullo, D.; Akocak, S.; Walker, E.A.; Supuran, C.T.; Ilies, M.A. Chem. Commun. 2014, 50, 5980; c) Abdelrahim, M.Y.; Tanc, M.; Winum, J.Y.; Supuran, C.T.; Barboiu, M. Chem. Commun. 2014, 50, 8043; d) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran, C.T. J. Med. Chem. 2000, 43, 4542. 21. Ferraroni, M.; Del Prete, S.; Vullo, D.; Capasso, C.; Supuran, C.T. Acta Crystall. D. Biol. Crystal. 2015, 71, 2449.
13
Carbonic anhydrase activators: Activation of the -carbonic anhydrase from Malassezia globosa with amines and amino acids Daniela Vullo, Sonia Del Prete, Clemente Capasso,* and Claudiu T. Supuran* OH
OH
O O
H
N
+ H
H
H O H H N
+
O
NH2
Arg51
O
H2 N
Asp49
H
H O
Cys47
- Zn S
2+
S
Cys106
N N H
His103
14
S0960-894X(16)30078-6 http://dx.doi.org/10.1016/j.bmcl.2016.01.078 BMCL 23543
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
4 January 2016 25 January 2016 28 January 2016
Please cite this article as: Vullo, D., Prete, S.D., Capasso, C., Supuran, C.T., Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.078
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Carbonic anhydrase activators: Activation of the -carbonic anhydrase from Malassezia globosa with amines and amino acids Daniela Vullo,a Sonia Del Prete, b,c Clemente Capasso,b* and Claudiu T. Supurana,c* a
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della
Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy. b
Istituto di Biochimica delle Proteine – CNR, Via P. Castellino 111, 80131 Napoli, Italy.
c
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm.
188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. The -carbonic anhydrase (CA, EC 4.2.1.1) from the dandruff producing fungus Malassezia globosa, MgCA, was investigated for its activation with amines and amino acids. MgCA was weakly activated by amino acids such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3 M) but more effectively activated by D-Phe, LDOPA, L-Trp, histamine, dopamine, pyridyl-alkylamines, and 4-(2-aminoethyl)-morpholine), with KAs of 5.82-10.9 M. The best activators were L-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81 M. This study may help a better understanding of the activation mechanisms of -CAs from pathogenic fungi as well as the design of tighter binding ligands for this enzyme which is a drug target for novel types of anti-dandruff agents.
Keywords: carbonic anhydrase; activator; amine; amino acid; dandruff; Malassezia globosa
______ * Corresponding authors. Tel/Fax: +39-0816132559. E-mail: [email protected] (Clemente Capasso); Tel/Fax: +39-055-4573729, E-mail:[email protected] (Claudiu T. Supuran).
1
Various species of the fungal pathogen Malassezia are the ethiologic agents of dandruff, a condition affecting billions of persons.1 Dandruff is triggered by several factors, including an increase in sebum production, irritation by pathogenic organisms (particularly fungi belonging to the above mentioned genus), individual susceptibility factors and stress. 1 The recent characterization of a carbonic anhydrase (CA, EC 4.2.1.1) belonging to the -class in Malassezia globosa.
1,2
MgCA, led us to initiate a systematic search for modulators of this enzyme activity, i.e., inhibitors and activators, which might show therapeutic potential. 2,3 Of the six independently-evolved (, , , , and η) CA classes discovered to date, only and -CAs were reported so far in fungi:2-5 basidiomycetes and hemiascomycetouos yeasts contain only -CAs, whereas the filamentous ascomycetes also possess -CAs in addition to the -class enzymes.2a Yet, the CAs play crucial functions in the life cycles of pathogenic fungi, among which CO2 sensing, by coupling with an adenylyl cyclase, which allows the pathogen to adapt to the dramatic changes in CO2 levels during colonization and infection of the human host and to fine-tune its virulence in the various physiologic conditions in which it grows. 3-5 Muhlschlegel’s group showed3a that inhibition with ethoxzolamide, a strong sulfonamide CA inhibitor, of the -CAs from fungal pathogenic species such as Candida albicans or Cryptococcus neoformans lead, in certain conditions, to the inhibition of the growth of the pathogen, whereas our group showed that inhibition of MgCA with various sulfonamide inhibitors has the same effect,2b leading to an impairment of the pathogen growth with fragmented fungal hyphae, a situation also observed after the treatment with ketoconazole, a clinically used antifungal drug acting through a different mechanism of action.2b CAs are present in many pathogenic organisms apart fungi, such as bacteria and protozoa, where they play crucial roles in the microbial virulence. 6,7 The CA distribution pattern in bacteria is very intriguing,as some bacteria encode CAs belonging just to one class, others from two and even three different genetic classes (i.e., α-, β- and γ-CAs), whereas the protozoa encode for α-, β- and ηclass enzymes, some of which were recently shown to be drug targets.7,8 Inhibition of CAs, mainly of the mammalian -class enzymes, was investigated in great detail with several clinically used CA inhibitors (CAIs) having applications as diuretics, antiglaucoma, antiobesity or anticancer agents/diagnostic tools. 6 The inhibition of fungal CAs, such as the Candia albicans, Candida glabrata, Cryptococcus neoformans and Saccharomyces cerevisiae enzymes with sulfonamides, carboxylates and dithiocarbamates was also investigated in the lat period,3,9,10 in the search of antifungals with a different mechanism of action, considering the serious drug resistance problems with the presently used clinical agents belonging to most antifungal classes.2,3,10 2
On the other hand, CA activators (CAAs), were less investigated in pathogenic organisms, as this class of enzyme modulators started to be investigated systematically for their interaction with mammalian -CAs only in the past years.11-14 Indeed, our group reported kinetic and X-ray crystallographic studies for the interaction of all human CA isoforms (hCA I – XV) with amino acid and amine activators, also unravelling the activation mechanism with this class of modulators of activity.11a-e,12-14 Although the mammalian enzymes CAAs do not have clinical applications for the moment, recent work from Muller’s group11f,g highlighted the important role that the CA activators play in modulating the calcification of the bone, opening interesting potential application for the modulators of CA activity for obtaining artificial tissues. Recently we have extended the CA activation studies to the -class enzymes from pathogenic fungi, reporting the first such studies for S. cerevisiae, ScCA15a (encoded by the NCE103 gene found in all basidiomycetes and hemiascomycetous yeasts),2 C. glabrata, CgCA,15b and C. albicans CaCA enzymes.15c These studies may bring interesting information regarding the physiologic roles of these enzymes, which seem to be involved in metabolic, biosynthetic processes (e.g. fatty acid biosynthesis) 16 apart the chemosensing roles mentioned above.4,5 All CA classes have a metal hydroxide cofactor within the active center, which is crucial for catalysis.2,3,6,7 The CA activation mechanism considers the rate-determining step of the CA catalytic cycle (for the hydration of CO2 to bicarbonate), which is the formation of the zinc hydroxide species of the enzyme (zinc is the prevalent metal ion in various CA classes, being the natural one in the fungal CAs).6a,c,11 This process occurs via the transfer of a proton from the Zn(II)-coordinated water molecule to the environment and is assisted by amino acid residues from the enzyme active site or by the binding of an activator molecule within the enzyme cavity.11-15 For the -CAs these phenomena are well understood, as many X-ray crystal structures of enzyme-activator adducts are available.11,12,14 Although few studies that have been published to date for other CA classes, there is evidence that activation mechanisms of the - and -CAs are similar.15 For example, the binding of the CA activator within the enzyme active site, which leads to the formation of an enzyme – activator complex seems to occur also in - and -CAs investigated to date.11-15 This adduct facilitates the shuttling of protons between the Zn(II) ion-coordinated water molecule and the environment, generating thus the nucleophilic zinc hydroxide, which is the catalytically active species of the enzymes.11-15 Here we report the first activation study of MgCA with a series of amines and amino acids (of types 1-19, Fig. 1), which were investigated earlier
11-14
for their interactions with mammalian
-CAs as well as more recently with the - and -class enzymes from the Archaea domain and from pathogenic fungi, respectively.15 This study may help a better understanding of the -CA 3
catalytic/activation mechanism, as the natural proton shuttling residue in this class of enzymes has not been yet identified, unlike the α-CAs for which it is known that a His residue placed in the middle of the active site cavity (His64, hCA I numbering) plays this function.6 O
O
O
H2N
H2N
OH
H2N
OH
OH OH
N N 1: L-His 2: D-His
3: L-Phe 4: D-Phe
5: L-DOPA 6: D-DOPA
O
O
O
H2N
H2N
OH
H2N
OH
OH
OH
N NH2
OH 9: L-Tyr 10: D-Tyr
7: L-Trp 8: D-Trp
N
11: 4-H2N-L-Phe
NH2
NH2
NH2
HO
N H
N H
OH
12 OH
14
13 H N
NH2 ( )n
15: n = 1 16: n = 2
HO X
OH H N
N NH2 17: X = NH 18: X = O
HO 19
Fig. 1: Structure of activators 1-19 investigated in the present study. Data of Table 117 show histamine to be an activator of MgCA as well as hCA II and β-class enzymes from other fungal pathogens, investigated earlier.15 At a concentration of 10 M, histamine enhanced kcat values for all the enzymes, whereas the KM values remain unchanged. According to previous studies, histamine is a millimolar activator for the -class enzyme (hCA II), with KA of 125 M,11 whereas it is a more effective activator for the fungal ones, such as the yeast ScCA (KA of 20.4 M) or C. glabrata CgCA enzymes (KA of 27.4 M).15 The results from the present study show that histamine has a higher affinity for MgCA with a KA of 10.9 ± 0.9 M (see discussion later in the text) compared to the other yeast/fungal enzymes. It is thus obvious that the activation mechanisms of the - and -CAs are likely to be similar, i.e., the CA activator enhances
4
kcat but has no influence on KM, facilitating the release of the proton from the water coordinated to the catalytic zinc ion. Table 1: Kinetic parameters for the activation of human hCA II (-class) and -class enzymes from S. cerevisiae (scCA),15a C. glabrata (CgCA),15b and M. globosa (MgCA) with histamine (Hst) 12, measured at 25 °C, pH 8.3 in 20 mM Tris buffer and 20 mM NaClO 4, for the CO2 hydration reaction, MgCA concentration in the assay system was of 12.1 nM. 16
Isozyme
kcat*
KM*
(kcat)Hst**
KA*** (M)
(s-1)
(mM)
(s-1)
Hst
hCA IIa
1.4x106
9.3
2.0x106
125
ScCAb
9.4 x105
9.5
19.6 x105
20.4
CgCAb
3.8 x105
7.9
10.7 x105
c
MgCA
(9.2±0.1)x10
5
11.1±0.1
(18.5 ±0.3) x10
27.4 5
10.9 ±0.9
_______________________________________________________________________________ * Observed catalytic rate without activator. KM values in the presence and the absence of activators were the same for the various CAs (data not shown). ** Observed catalytic rate in the presence of 10 M activator. *** The activation constant (KA) for each enzyme was obtained by fitting the observed catalytic enhancements as a function of the activator concentration. 16 Mean from at least three determinations by a stopped-flow, CO2 hydrase method.16 Standard errors were in the range of 5-10 % of the reported values. a
Human recombinant enzyme, data from ref. 14
b
Yeast/fungal recombinant enzymes data from ref. 15a,b
c
This work. Mean ± standard error (from 3 different assays).
Data of Table 2 show that all amino acids and amines 1-19, which have been investigated in this study, act as CAAs against the fungal enzyme MgCA. However, the activation profiles of these compounds against MgCA are different from other recently investigated - and -class enzymes, such as hCA II, ScCA and CgCA,11,15 which were included in Table 2 for comparison. The following structure activity relationship (SAR) can be observed for the activation of MgCA with compounds 1-19:17-20 (i) Several amino acids, such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr, 4-amino-Lphenylalanine, as well as serotonin 14, showed activation constants in the range of 12.5 – 29.3 M, therefore they are considered as moderate MgCA activators. However, it may be observed that most of these derivatives (except L-Phe, L-/D-Tyr and D-Trp) are much weaker activators of the C.
5
glabrata enzyme CgCA. ScCA on the other hand has completely different activation profiles with these derivatives,being poorly activated by all amino acids (but slightly more susceptible to activation by serotonin). hCA II is highly activated by most amino acids, sometimes with activation constants in the nanomolar range 11-14 (e.g., L-/D-Phe; L-/D-Tyr), see Table 2. Table 2: Activation constants of hCA II (cytosolic α-isozyme), yeast -CAs from S. cerevisiae (ScCA), C. glabrata (CgCA) and MgCA (Malassezia globosa) with amino acids and amines 1 – 19. Activation data of hCA II, ScCA and CgCA with these compounds are retrieved from refs.14,15a,b No.
Compound
KA (M)* hCA IIa
ScCAb
CgCAc
MgCAd
1
L-His
10.9
82
37.0
29.3 ± 1.2
2
D-His
43
85
21.2
18.1 ± 0.9
3
L-Phe
0.013
86
24.1
34.1 ± 2.7
4
D-Phe
0.035
86
15.7
10.7 ± 0.8
5
L-DOPA
11.4
90
23.3
8.31 ± 0.6
6
D-DOPA
7.8
89
15.1
13.7 ± 1.1
7
L-Trp
27
91
22.8
10.1 ± 0.6
8
D-Trp
12
90
12.1
12.5 ± 1.2
9
L-Tyr
0.011
85
9.5
15.7 ± 1.0
10
D-Tyr
0.058
84
7.1
25.1 ± 1.9
11
4-H2N-L-Phe
0.15
21.3
31.6
13.4 ± 0.8
12
Histamine
125
20.4
27.4
10.9 ± 0.9
13
Dopamine
9.2
13.1
27.6
9.43± 0.7
14
Serotonin
50
15.0
16.7
14.2 ± 1.3
15
2-Pyridyl-methylamine
34
16.2
15.0
6.12 ± 0.3
16
2-(2-Aminoethyl)pyridine 15
11.2
16.3
7.30 ± 0.3
17
1-(2-Aminoethyl)-piperazine
9.3
14.9
0.81 ± 0.07
18
4-(2-Aminoethyl)-morpholine
10.2
10.1
5.82 ± 0.4
19
L-Adrenaline
0.95
10.8
0.72 ± 0.05
2.3 0.19
96
* Mean from three determinations by a stopped-flow, CO2 hydrase method.25 Standard errors were in the range of 5-10 % of the reported values for the activation of hCA II, ScCA and CgCA. 21,23,24 a
Human recombinant isozyme, from ref. 21
b
Recombinant archaeal enzyme, from ref. 23
c
Recombinant yeast enzyme, from ref. 24
d
Recombinant yeast enzyme, this study. Mean ± standard error (from 3 different assays). 6
(ii) A second group of derivatives, including D-Phe, L-DOPA, L-Trp, as well as amines 12, 13, 15, 16 and 18, acted as more effective MgCA activators when compared to the compounds discussed above, with activation constants ranging between 5.82 – 10.9 M. Thus, it may be observed that the amines were generally better MgCA activators compared to the carboxylic acids with which they are structurally related (compare Histamine with L/D-His, or dopamine with L-/DDOPA, for example). The heterocylic amines incorporating pyridyl, piperazine, and morpholine rings were more effective MgCA activators compared to histamine, dopamine or serotonin. (iii) The most potent MgCA activators were L-adrenaline 19 and 1-(2-aminoethyl)piperazine 17, with activation constants of 0.72-0.81 M. Thus, small structural modifications in the scaffold of the amine activator (e.g., compare dopamine 13 with L-adrenaline 19) lead to drastic changes of the activating effect, with 19 being 13-times a better activator compared to 13.
OH
OH
O O
H
N
+ H
H
H O H H N
+
O
NH2
Arg51
O
H2 N
Asp49
H
H O
Cys47
- Zn S
2+
S
Cys106
N N H
His103
Fig. 2: Proposed schematic interactions between an activator (L-DOPA, 5) and the MgCA active site. The Zn(II) ion is coordinated by two Cys and one His residue, and by a water molecule (M. globosa CA numbering of amino acid residues).2b Both the amino and carboxylate moieties of the activator 5 can participate in hydrogen bonds with amino acid residues in the neighborhood of the metal ion center and facilitate the transfer of the proton from the zinc-bound water to the 7
environment with generation of the zinc hydroxide species of the enzyme. A network of hydrogen bonds between the bridging water molecule interacting with the activator and the Asp49 - Arg51 catalytic dyad and zinc coordinated water has been hypothesized here, but the real number of water molecules participating in the activation process is unknown. In hCA II – activator crystal structures it has been shown to be between 1 and 3 water molecules. 11,14 A possible activation mechanism of the M. globosa -CAs by L-DOPA 5 is depicted schematically in Fig. 2. As for other -CAs, the catalytic Zn(II) ion in the MgCA active site is coordinated by two Cys and one His residues (Cys47, His103 and Cys106, MgCA numbering system).2b A second pair of conserved amino acid residues in all sequenced -CAs known to date,2-7 is constituted by the dyad Asp49 – Arg51 (M. globosa CA numbering). These amino acids are close2-4 to the zinc-bound water molecule, which is the fourth Zn(II) ligand in this type of open active site -CAs,2 and participate in a network of hydrogen bonds, which probably assist the water deprotonation and the formation of the nucleophilic, zinc hydroxide species of the enzyme. 8,10 The active site channel of -CAs (as exemplified by the X-ray crystal structure of the C. neoformans enzyme Can23a or Vibrio cholerae
21
enzymes) is a channel which can accommodate elongated
molecules such as the aromatic amino acids/amines which have been investigated here. Thus, we hypothesize that the activator (such as L-DOPA 5) may bind closely to the active site channel, establishing supplementary hydrogen bonds with the polar moieties of amino acid residues such as the catalytic dyad mentioned above, or with the zinc-bound water molecule (directly or through a network of several other water molecules, as demonstrated for the interaction of -CAs with this type of activator)11,14 and thus assist water deprotonation and facilitate the catalytic turnover. Indeed, both the amino or carboxyl moieties of the activators can establish hydrogen bonds with these structural elements, due to the presence of many heteroatoms in their molecules. Fig. 2 shows schematically a putative binding mode of L-DOPA 2 within the active site of MgCA. This hypothesis should be confirmed by X-ray crystallography, however the structure of MgCA has not yet been resolved. In conclusion, we report the first activation study of the -CA from the fungal pathogen M. globosa with amines and amino acids. MgCA was weakly activated by amino acids such as L-/DHis, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3 M) but more effectively activated by D-Phe, L-DOPA, L-Trp, histamine, dopamine, pyridyl-alkylamines, and 4(2-aminoethyl)-morpholine), with KAs of 5.82-10.9 M. The best activators were L-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81 M. This study may help a better understanding of the activation mechanisms of -CAs from pathogenic fungi as well as the 8
design of tighter binding ligands for this enzyme which is a drug target for novel types of antidandruff agents. Acknowledgments. This research was financed in part by a grant of the 7th Framework Programme of the European Union (Dynano project).
9
References and Notes
1. a) Clavaud, C.; Jourdain, R.; Bar-Hen, A.; Tichit, M.; Bouchier, C.; Pouradier, F.; El Rawadi, C.; Guillot, J.; Ménard-Szczebara, F.; Breton, L.; Latgé, J.P.; Mouyna, I. PLoS One. 2013, 8, e58203; b) Donnarumma, G.; Perfetto, B.; Paoletti, I.; Oliviero, G.; Clavaud, C.; Del Bufalo, A.; Guéniche, A.; Jourdain, R.; Tufano, M.A.; Breton, L. Arch. Dermatol. Res. 2014, 306, 763. 2. a) Elleuche, S.; Poggeler, S. Curr. Genet. 2009, 55, 211; b) Hewitson, K.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C.T. J. Med. Chem. 2012, 55, 3513; c) Del Prete, S.; De Luca, V.; Vullo, D.; Osman, S.M.; AlOthman, Z.; Carginale, V.; Supuran, C.T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2016, 31, in press (doi: 10.3109/14756366.2015.1102137); d) Del Prete, S.; Vullo, D.; Osman, S.M.; AlOthman, Z.; Capasso, C.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2015, 25, 5194. 3. a) Schlicker, C.; Hall, R.A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C.T.; Muhlschlegel, F.A.; Steegborn, C. J. Mol. Biol. 2009, 385, 1207; b) Hall, R.A.; Mühlschlegel, F.A. Fungal and nematode carbonic anhydrases: Their inhibition in drug design. In Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications, Supuran, C.T.; Winum, J.Y. Eds., John Wiley & Sons, Hoboken, 2009, pp. 301 – 322. 4. a) Klengel, T.; Liang, W.J.; Chaloupka, J.; Ruoff, C.; Schropel, K.; Naglik, J.R.; Eckert, S.E.; Mogensen, E.G.; Haynes, K.; Tuite, M.F.; Levin, L.R.; Buck, J.; Mühlschlegel, F.A. Curr. Biol. 2005, 15, 2021; b) Bahn, Y.S.; Cox, G.M:, Perfect, J.R.; Heitman, J. Curr. Biol. 2005, 15, 2013. 5. a) Mogensen, E.G.; Janbon, G.; Chaloupka, J.; Steegborn, C.; Fu, M.S.; Moyrand, F.; Klengel, T.; Pearson, D.S.; Geeves, M.A., Buck, J.; Levin, L.R.; Mühlschlegel, F.A. Eukaryot. Cell 2006, 5, 103; b) Bahn, Y.S.; Mühlschlegel, F.A. Curr. Opin. Microbiol. 2006, 9, 572. 6. a) Supuran, C.T. Nat. Rev. Drug. Discov. 2008, 7, 168; b) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2016, 31, in press (doi: 10.3109/ 14756366.2015.1122001); c) Supuran, C.T. Expert Opin. Ther. Pat.2013, 23, 677; c) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; d) Supuran, C.T. Curr. Top. Med. Chem. 2007, 7, 825; e) Supuran, C.T.; Scozzafava, A.; Mastrolorenzo, A. Expert Opin. Ther. Pat. 2001, 11, 221. 7. a) Tripp, B.C.; Smith, K.S.; Ferry, J.G. J. Biol. Chem. 2001, 276, 48615; b) Smith, K.S.; Ferry, J.G. FEMS Microbiol. Rev. 2000, 24, 335; c) Zimmerman, S.A.; Ferry, J.G.; Supuran, C.T. Curr. Top. Med. Chem. 2007, 7, 901; d) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; e) Del Prete, S.; Vullo, D.; Fisher, G.M.; Andrews, K.T.; Poulsen, S.A.; Capasso, C.; Supuran, C.T. Bioorg. Med. Chem. Lett.2014, 24, 4389; f) Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.; Scozzafava, A.; Dedhar, S.; Winum, J.Y.; Supuran, C. T. J. Med. Chem. 2011, 54, 8271.
10
8. a) Supuran, C. T. Front Pharmacol 2011, 2, 34; b) Capasso, C.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379; c) Capasso, C.; Supuran, C.T. Expert Opin. Ther. Targets 2015, 19, 1689; d) Supuran, C.T.; Capasso, C. Expert Opin. Ther. Targets 2015, 19, 551; d) Capasso, C.; Supuran, C.T. Expert Opin. Ther. Pat. 2013, 23, 693; e) Capasso, C.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2015, 30, 325; f) Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C.T. Curr. Pharm. Des. 2008, 14, 622. 9. a) Aguilera, J.; Van Dijken, J. P.; De Winde, J. H.; Pronk, J. T., Biochem. J. 2005, 391, 311; b) Aguilera, J.; Petit, T.; de Winde, J.; Pronk, J. T. FEMS Yeast Res. 2005, 5, 579. 10. a) Innocenti, A.; Mühlschlegel, F.A.; Hall, R.A.; Steegborn, C.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2008, 18, 5066; b) Innocenti, A.; Hall, R.A.; Schlicker, C.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. 2009, 17, 2654; c) Innocenti, A.; Hall, R.A.; Schlicker, C.; Scozzafava, A.; Steegborn, C.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. 2009, 17, 4503; d) Innocenti, A.; Leewattanapasuk, W.; Mühlschlegel, F.A.; Mastrolorenzo, A.; Supuran, C.T.. Bioorg. Med. Chem. Lett. 2009, 19, 4802; e) De Simone, G.; Di Fiore, A.; Supuran, C.T. Curr. Pharm. Des. 2008, 14, 655. 11. a) Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C.T. Biochemistry, 1997, 36, 10384; b) Temperini, C.; Scozzafava, A.; Supuran, C.T. Curr. Pharm. Des. 2008, 14, 708; c) Ilies, M.; Scozzafava, A.; Supuran, C.T. Carbonic anhydrase activators. In Carbonic anhydrase - Its inhibitors and activators, Supuran, C.T.; Scozzafava, A.; Conway J. Eds., CRC Press, Boca Raton (FL), USA, 2004, pp. 317-352; d) Supuran, C.T.; Scozzafava, A. Activation of carbonic anhydrase isozymes. In The Carbonic Anhydrases - New Horizons, Chegwidden, W.R.; Carter, N.; Edwards, Y. Eds.; Birkhauser Verlag: Basel, Switzerland, 2000, pp. 197-219; e) Supuran, C.T. Therapy, 2007, 4, 355; f) Wang, X.; Schröder, H.C.; Müller, W.E. Int. Rev. Cell. Mol. Biol. 2014, 313, 27; g) Wang, X.; Schröder, H.C.; Schlossmacher, U.; Neufurth, M.; Feng, Q.; Diehl-Seifert, B.; Müller, W.E. Calcif. Tissue Int. 2014, 94, 495. 12. a) Scozzafava, A.; Supuran, C.T. J. Med. Chem. 2002, 45, 284; b) Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2002, 12, 1177; c) Ilies, M.; Banciu, M.D.; Ilies, M.A.; Scozzafava, A.; Caproiu, M.T.; Supuran, C.T. J. Med. Chem. 2002, 45, 504; d) Scozzafava, A.; Iorga, B.; Supuran, C.T. J. Enzyme Inhib. 2000, 15, 139; e) Temperini, C.; Scozzafava, A.; Vullo, D.; Supuran, C.T. J. Med. Chem. 2006, 49, 3019. 13. a) Parkkila, S.; Vullo, D.; Puccetti, L.; Parkkila, A.K.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem Lett. 2006, 16, 3955; b) Vullo, D.; Nishimori, I.; Innocenti, A.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2007, 17, 1336; c) Clare, B.W.; Supuran, C.T. J. Pharm.
11
Sci. 1994, 83, 768; d) Saada, M.C.; Montero, J.L.; Winum, J.Y.; Vullo, D.; Scozzafava, A.; Supuran, C.T. J. Med. Chem. 2011, 54, 1170. 14. a) Temperini, C.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2007, 17, 628; b) Vullo, D.; Nishimori, I.; Innocenti, A.; Scozzafava, A.; C.T. Supuran, Bioorg. Med. Chem. Lett. 2007, 17, 1336; c) Vullo, D.; Innocenti, A.; Nishimori, I.; Scozzafava, A.; Kaila, K.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2007, 17, 4107; d) Pastorekova, S.; Vullo, D.; Nishimori, I.; Scozzafava, A.; Pastorek, J.; Supuran, C.T. Bioorg. Med. Chem. 2008, 16, 3530. 15. a) Isik, S.; Kockar, F.; Aydin, M.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A..; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2009, 19, 1662; b) Innocenti, A.; Leewattanapasuk, W.; Manole, G.; Scozzafava, A.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2010, 20, 1701; c) Innocenti, A.; Hall, R.A.; Scozzafava, A.; Mühlschlegel, F.A.; Supuran, C.T. Bioorg. Med. Chem. 2010, 18, 1034; d) Innocenti, A.; Zimmerman, S.A.; Scozzafava, A.; Ferry, J.G.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2008, 18, 6194; e) Supuran, C.T.; Clare, B.W. Eur. J. Med. Chem. 1999, 34, 41. 16. a) Kim, M.S.; Ko, Y.J.; Maeng, S.; Floyd, A.; Heitman, J. Bahn,; Y.S. Genetics 2010, 185, 1207; b) Lehneck, R.; Pöggeler, S. Appl. Microbiol. Biotechnol. 2014, 98, 8433; c) Lehneck, R.; Neumann, P.; Vullo, D.; Elleuche, S.; Supuran, C.T.; Ficner, R.; Pöggeler, S. FEBS J.2014, 281, 1759; d) Mastrolorenzo, A.; Rusconi, S.; Scozzafava, A.; Barbaro, G.; Supuran, C.T. Curr. Med. Chem. 2007, 14, 2734; e) Bayram, E.; Senturk, M.; Kufrevioglu, O.I.; Supuran, C.T. Bioorg. Med. Chem. 2008, 16, 9101. 17. Khalifah, R.G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-flow instrument was used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, 10 – 20 mM Hepes (pH 7.5, for α-CAs) or Tris (pH 8.3, for β-CAs) as buffers, and 20 mM Na2SO4 or 20 mM NaClO4 (for maintaining constant the ionic strength), following the CA-catalyzed CO2 hydration reaction for a period of 10 s at 25°C. The CO 2 concentrations ranged from 0.17 to 17 mM for the determination of the kinetic parameters and activation constants. For each activator at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activators 1-19 (10 mM) were prepared in distilled-deionized water and dilutions up to 0.001 M were done thereafter with distilled-deionized water. Activator and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-A complex. The activation constant (KA), defined similarly with the inhibition
12
constant KI,18-20 can be obtained by considering the classical Michaelis-Menten equation (equation 1), which has been fitted by non-linear least squeares by using PRISM 3: v = vmax /1 + KM /[S]x(1 + [A]f/KA)
(1)
where [A] f is the free concentration of activator. Working at substrate concentrations considerably lower than K M ([S] << KM), and considering that [A] f can be represented in the form of the total concentration of the enzyme ([E] t) and activator ([A]t), the obtained competitive steady-state equation for determining the activation constant is given by eq. 2:18-20 v = v0 . KA /KA + ([A]t – 0.5 ([A]t + [E]t + KA) – ([A]t + [E]t + KA)2 – 4[A]t.[E]t)1/2 (2) where v0 represents the initial velocity of the enzyme-catalyzed reaction in the absence of activator.18-20 The concentration of the enzymes in the assay system were in the range of 10-14 nM. 18. a) Temperini, C.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2006, 16, 5152; b) Temperini, C.; Innocenti, A.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. 2008, 16, 8373; c) Vullo, D.; Voipio, J.; Innocenti, A.; Rivera, C.; Ranki, H.; Scozzafava, A.; Kaila, K.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2005, 15, 971; d) Nishimori, I.; Minakuchi, T.; Morimoto, K.; Sano, S.; Onishi, S.; Takeuchi, H.; Vullo, D.; Scozzafava, A.; Supuran, C.T. J. Med. Chem. 2006, 49, 2117. 19. a) Abdo, M.R.; Vullo, D.; Saada, M.C.; Montero, J.L.; Scozzafava, A.; Winum, J.Y.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2009, 19, 2440; b) Saada, M.C.; Montero, J.L.; Vullo, D.; Scozzafava, A.; Winum, J.Y.; Supuran, C.T. Bioorg. Med. Chem. 2014, 22, 4752; c) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C.T. J. Med.Chem. 2010, 53, 5511. 20. a) Dave, K.; Scozzafava, A.; Vullo, D.; Supuran, C.T.; Ilies, M.A. Org. Biomol. Chem. 2011, 9, 2790; b) Draghici, B.; Vullo, D.; Akocak, S.; Walker, E.A.; Supuran, C.T.; Ilies, M.A. Chem. Commun. 2014, 50, 5980; c) Abdelrahim, M.Y.; Tanc, M.; Winum, J.Y.; Supuran, C.T.; Barboiu, M. Chem. Commun. 2014, 50, 8043; d) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran, C.T. J. Med. Chem. 2000, 43, 4542. 21. Ferraroni, M.; Del Prete, S.; Vullo, D.; Capasso, C.; Supuran, C.T. Acta Crystall. D. Biol. Crystal. 2015, 71, 2449.
13
Carbonic anhydrase activators: Activation of the -carbonic anhydrase from Malassezia globosa with amines and amino acids Daniela Vullo, Sonia Del Prete, Clemente Capasso,* and Claudiu T. Supuran* OH
OH
O O
H
N
+ H
H
H O H H N
+
O
NH2
Arg51
O
H2 N
Asp49
H
H O
Cys47
- Zn S
2+
S
Cys106
N N H
His103
14