European Journal of Medicinal Chemistry 90 (2015) 577e582
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, anticandidal activity of N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic amide derivatives e Novel inhibitors of glucosamine-6-phosphate synthase Dorota Pawlak, Magdalena Stolarska, Marek Wojciechowski, Ryszard Andruszkiewicz* sk University of Technology, Gdan sk, Poland Department of Pharmaceutical Technology and Biochemistry, Gdan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2014 Received in revised form 3 December 2014 Accepted 4 December 2014 Available online 5 December 2014
Novel FMDP amides 4e6 have been synthesized and tested against Candida strains. The anticandidal activity has been confined only to Candida albicans. Anticandidal activity of the tested amides has correlated with their inhibitory activity of glucosamine-6-phosphate synthase in cell free extract from C. albicans. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Glucosamine-6-phosphate synthase inhibitors Antifungal activity Molecular modeling FMDP analogs
1. Introduction In recent years there has been a significant increase in the number of systemic fungal infections [1]. In many cases these infections lead to a serious diseases of internal organs such as the liver or lungs. Currently used chemotherapeutics in the antifungal therapy such as azoles, only exert fungistatic activity, therefore the incidence of azole-resistant fungal strains are still increasing [2]. On the other hand the most effective antifungal antibiotic such as amphotericin B displays a number of side effects. Toxicity, resistance phenomenon, low and narrow antifungal activity of known antifungal agents prompted the search for a new chemical compounds that could be used in the treatment of fungal infections. Glucosamine-6-phosphate (GlcN-6-P) synthase, has been proposed as one of the most perspective molecular targets for antifungal and antimicrobial therapy [3]. The enzyme is involved in the synthesis pathway of the cell wall. This enzyme catalyzes the transfer of an amino group of L-glutamine to D-fructose-6-phosphate with formation of D-glucosamine-6-phosphate which is a substrate for the
* Corresponding author. Department of Pharmaceutical Technology and sk University of Technology, 11/12 Narutowicza Street, 80-233 Biochemistry, Gdan sk, Poland. Gdan E-mail address: [email protected] (R. Andruszkiewicz). http://dx.doi.org/10.1016/j.ejmech.2014.12.007 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
synthesis of macromolecules containing chitin and mannoproteins in fungi, peptidoglycan and lipopolysaccharide of bacteria, as well as glycoproteins and glycolipids in higher organisms [4,5]. Inactivation of the enzyme in microbial cells leads to inhibition of cell wall synthesis and, consequently, to lysis of the cells without negative effects for mammalian cells. Additionally, due to participation of GlcN-6-P synthase in the biosynthesis of amino sugars, the enzyme may be involved into the regulation of blood glucose levels. Therefore, specific inhibitors of GlcN-6-P synthase activity may be also regarded as new therapeutic agent for type II diabetes [6]. A large number of compounds have showed the ability to inactivate GlcN-6-P synthase and the most effective ones, have been found to be the analogs of L-glutamine, the enzyme substrate. N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (FMDP) is one of the strongest and the most specific inhibitors of GlcN-6-P synthase. The polar character of the molecule prevents its penetration into cells by free diffusion, therefore FMDP itself, showed almost no activity when tested on fungal cell. Much efforts have been devoted to improve FMDP antifungal activity. The most successful approach based on the incorporation of FMDP molecule into short peptides [7]. These peptides were transported into cells via peptide permeases, then hydrolyzed with cytoplasmic peptides with the release of FMDP inhibitor inside the
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inhibitory potency against glucosamine-6-phosphate synthase when compared to the unmodified compound FMDP. There is a correlation between the size of amide function and ability to inhibit the enzyme. The most substituted, tertiary amide of FMDP (6) displayed the highest IC50 value.
cell [8]. Although a number of FMDP peptides exerted good activity when tested in vitro a rapid formation of FMDP peptides-resistant strains of Candida albicans may hamper the future utility of these agents. In order to overcome this problem, FMDP molecule has been modified by construction of more lipophilic derivatives of FMDP, e.g. by acylating its N-terminal amino group, or esterification its carboxyl group thus increasing their transport into cell by free diffusion [9]. Latent derivatives of FMDP were also synthesized and tested [10]. Their antifungal activity however was not acceptable at the present stage of development. A series of FMDP tertiary and secondary amides with improved lipophilicity, containing four to seven carbon atoms into alkyl chains, showed however, weak inhibitory activity towards fungal GlcN-6-P synthase, and relatively poor activity against fungal species. Development of inhibitor modified at the carboxyl group of with enhanced lipophilicity may still be solution to the GlcN-6-P synthase inhibitor transport problem. Thus we have decided to explore the carboxyl group for the construction of FMDP derivatives with GlcN-6-P synthase inhibitory activity and antifungal activity. In this communication we report the design and synthesis of three novel inhibitors of GlcN-6-P synthase, i.e. the primary amide, N-methyl and N,N-dimethylamide FMDP, evaluation of anticandidal activity and inhibitory properties toward fungal GlcN-6-P synthase (Fig. 1). Since glutamine binding site of the GlcN-6-P synthase is covered by so called Q-loop and thus does not have much room for large substituents, we first investigated by molecular modeling methods, how substitution of the charged carboxyl group with neutral and bulky N-methyl and N,N-dimethylamide affects the ligand interaction with the receptor.
Data presented in Table 1 indicate that the new compounds exhibit no activity against a series of antibacterial species, including Gram-negative and Gram-positive ones. Candida glabrata and Candida parapsilosis strains seems to be insensitive to the action of novel compounds. The activity of novel compounds is only confined to the yeast of C. albicans ATCC 10231. All compounds, however, have displayed much better activity against C. albicans than FMDP itself. The results also demonstrate that anticandidal activity strongly depends on the number of substituents at nitrogen atom of amide group. Compound 4 - primary amide, has shown to be the strongest inhibitor of the fungal cells growth. The in vitro anticandidal activity of new compound is also correlated with the GlcN-6-P synthase inhibitory properties. Compound 6 displayed the lowest inhibitory activity against GlcN6-P synthase and the poorest in vitro activity against C. albicans. FMDP itself showed the best enzyme inhibitory activity and the lack of antifungal activity which can be attributed to the polar character of FMDP. It can be concluded that the higher lipophilicity of FMDP amides influenced their antifungal activity. The research of the antimicrobial and inhibition activity of the newly obtained compounds was completed by the results of molecular modeling method.
2. Results and discussion
2.4. Membrane affinity
2.1. Synthesis
The ability to diffuse through biological membrane for new derivatives and unmodified inhibitor was investigated using a HPLC column mimicking the lipidic environment found in cell membrane - IAM (immobilized artificial membrane) PC DD2 [13]. The retention times measured for the examined compounds and affinity to the biological membrane expressed as log k0 IAM values may be approximately used as a measure of their lipophilic properties (Table 2). This data present that the log k’IAM values increase with the number of methyl group at the nitrogen atom. Despite the fact that the synthesized derivatives (4e6) have rather low affinity to the membrane, their affinity however, is much better than in the case of FMDP molecule itself. The affinity to the biological membrane depends not only on the compound lipophilicity but also on number of hydrogen bonds, electrostatic interactions and sterical effects as well. Therefore, the log k0 IAM values reflects rather membrane affinity than lipophilic properties of the compound.
The synthesis (Scheme 1) started with N2-tert-butoxycarbonylN -(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid prepared by known procedure [11]. The amide, N-methylamide and N,Ndimethylamide of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diamiopropanoic acid (1e3) were obtained using mixed anhydride method in good yields as crystalline compounds. Deprotection of the Boc groups with 4 M HCl in dioxane afforded compounds 4e6 as amorphous hydrochloride salts in acceptable yields. All new compound were fully characterized by 1H NMR and mass spectrum analyses. 3
2.2. Glucosamine-6-phosphate synthase inhibition studies The new synthesized compounds as well as the parent inhibitor FMDP were tested as inhibitors of purified C. albicans GlcN-6-P synthase overproduced by Escherichia coli [12]. Their ability to inhibit this enzyme was measured by determining a concentration which caused 50% inhibition of the enzyme. The results are presented in Table 2. All the compounds (4e6) showed lower
Fig. 1. Structures of new inhibitors.
2.3. Antimicrobial activity
2.5. Molecular modeling The docking results of the primary amide derivative of FMDP to GlcN-6-P synthase glutamine binding site were very promising. The lowest energy and the most abundant ligand pose (28 results out of 50) fits into the binding pocket very well resembling the pattern of crucial interactions of the crystal ligand (HGA) (Fig. 2) [14]. Unmodified free amine group recreates the interactions of the respective group of the HGA (and native ligand e glutamine) with Gly99, Thr76 and Asp123. Despite the fact that the negatively charged carboxyl group, present in FMDP and all other inhibitors known so far, is missing, the amide moiety introduced in the designed inhibitors is still able to partially recreate the network of substrate's favorable interactions with the receptor and also form new ones. Namely the CO moiety of the amide mimics the
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Scheme 1. Syntheses of new compound (4e6). a) N-methylmorpholine, isobutyl chloroformate; b) 40% RH(aq) (R ¼ NH2,NHCH3,N(CH3)2); c) 4 M HCl/Dioxane.
interactions of the carboxyl group with sidechains of Arg73 and His86, replacing the salt bridge with strong hydrogen bonds (OeHN distances are 1.87 and 1.88 respectively). On the other hand it turns out that the NH2 moiety of the amide, that is not present neither in the crystal ligand nor in the enzyme's natural substrate, forms weak hydrogen bonds to Asp123 side chain's carboxyl group and His77 backbone CO group (the NHeO distances are 2.18 and 2.13 respectively). All these aminoacids are essential for GlcN-6-P synthase interactions with ligands and are conserved in the sequences of this enzyme from all organisms including C. albicans and E. coli [5]. Surprisingly, N-methylamide derivative of FMDP also fits well into the glutamine binding pocket of GlcN-6-P synthase, despite the bulky methyl group replacing one of the hydrogen atoms (Fig. 2). It turned out that the increased volume of the secondary amide moiety can be compensated by only small shift of the ligand while most of its essential interactions with the receptor can be preserved. The CO moiety of the secondary amide still forms the hydrogen bonds with Arg73 guanidine moiety and His86 side chain (OeHN distances 1.92 and 2.09 respectively) while remaining NH group of the amide forms weak hydrogen bonds to Asp123 sidechain's carboxyl group (the OeHN distance 2.01). Interestingly missing, due to the substitution of the second hydrogen with methyl, hydrogen bond to His77 is replaced with van der Waals interactions of the methyl group with Thr122 aliphatic part of the side chain (CeCG2 distance 3.06). Thus binding of the N-methylamide derivative of FMDP is only lowered with respect to the primary amide derivative. It is also reflected by the predicted binding free energy of the former increased by 1.5 kcal/mol. Docking of N,N-dimethylamide derivative of FMDP did not give consistent results. It seems that the tertiary amide bearing two bulky methyl groups prevents the ligand from binding in the proper orientation since there is not enough room inside the glutamine binding site. All predicted ligand poses were found to be rotated with respect to the crystal ligand and natural substrate, with N,Ndimethylamide moiety positioned outside of the binding pocket. 3. Conclusions We have synthesized three novel FMDP amides as new inhibitors of GlcN-6-P synthase. Primary amide of FMDP shows the highest anticandidal activity out of the amides tested, the activity
however was limited only to C. albicans strain. N-methyl amide of FMDP as well as its N,N-dimethylamide displayed significantly lower activity against C. albicans. The anticandidal activity of the synthesized compounds is also correlated with their inhibitory activity towards GlcN-6-P synthase. 4. Experimental 4.1. Chemistry Thin layer chromatography (TLC) was performed using Merck aluminum plates (Kieselgel 60 F254) and visualized by ultraviolet (UV) light. Column chromatography was carried out using silica gel (70e230 mesh) (Merck). MS spectrum was recorded on Agilent Technologies 6540 UDH Accurate-Mass Q-TOF. 1H NMR spectra were recorded on Gemini Varian spectrometer operating at 200 MHz using DMSO and D2O as solvents. 4.2. General procedure for the preparation of amide of N2-tertbutoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic acid 1e3 The N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic acid (FMDP) (0.95 g, 3 mmol) was dissolved in dry THF, cooled to a temperature of 5 C, then (0.33 mL, 3 mmol) N-methylmorpholine and (0.94 mL, 3 mmol) isobutyl chloroformate were added successively. After 10 min an equimolar amount of the corresponding amine solution in water [NH3 (25% aq), CH3NH2 (40% aq), NH(CH3)2 (40% aq)] was added. After 24 h of reaction, the solvent was evaporated to dryness and the residue was dissolved in ethyl acetate. The organic layer was washed with 1 M NaHSO4 solution, water, 1 M NaHCO3 solution, than the organic solvent was removed in vacuo to give the title compound as white solid. 4.2.1. Amide of N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)(S)-2,3-diaminopropanoic acid 1 Yield 0.810 g, 86%. M.p. 136e138 C. 1H NMR (200 MHz, DMSO): d 1.37 (s, 9H. C(CH3)3), 3.55 (m, 2H, CH2NH), 3.75 (s, 3H, COOCH3), 4.1 (m, 1H, CHCH2), 6.7 (d, 1H, CHNH), 6.8 (ABq, 2H, J ¼ 14.12, CH] CH), 7.25 (s, 1H, C(O)NH), 7.36 (s, 1H, C(O)NH), 8.6 (m, 1H, CH2NHCO).
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Table 1 Antimicrobial activity of compounds 4e6 expressed as MIC (mg/mL) and compared with FMDP. Co.
R]
C. albicans ATCC 10231
C. glabra ta DSM 11226
C. parapsilosis DSM S. aureus ATCC 5784 29213
S. epidermidis PCM E. coli PCM 2118 2560
B. subtilis ATCC9372
P aeruginosa ATCC 2756
4 5 6 FMDP NvaFMDP
NH2 NHCH3 N(CH3)2 e e
4 32 512 >2000 0.25
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 128
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 >1024
Table 2 Inhibitory data for compound 4e6 and FMDP in respect to C. albicans GlcN-6-P synthase and affinity to biological membranes. Compound
Inhibition IC50(mM)
4 5 6 FMDP
80.70 127.05 2894.01 4.03
± ± ± ±
5.90 12.57 331.74 0.26
Log k'IAM 0.362 0.193 0.118 0.812
>1024 >1024 >1024 >2000 8
4.3. General method for the preparation of hydrochloride of N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid amide 4e6 The tert-butoxycarbonyl protecting group was removed by treating of the resulting compound (1e3) with 4 M HCl in dioxane (5 mL) for 2.5 h. Then the solvent was removed in vacuo. The residue was triturated with diethyl ether and the precipitate was filtered off and dried in vacuo over KOH.
Fig. 2. Molecular modeling. Best scoring poses of the designed ligands bound to the 1gms receptor. Ligands are shown as thick sticks models, while sidechains of essential active site residues are drawn as thin sticks. Hydrogen bonds between ligands a the receptor are marked with blue dashed lines. Left e best scoring pose of the primary amide derivative of FMDP. Right e best scoring pose of the secondary amide derivative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4.2.2. N-methylamide of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid 2 Yield 0.88 g, 66%. M.p. 115 C. 1H NMR (200 MHz, DMSO): d 1.4 (s, 9H, C(CH3)3); 3.35 (s, 3H, NHCH3); 3.4 (m, 2H, CHCH2); 3.75 (s, 3H, OCH3); 4.1 (m, 1H, CHCH2); 6.8 (ABq, 2H, J ¼ 15.56, CH]CH); 6.84 (d, 1H, J ¼ 8.19, NHCH); 7.85 (m, 1H, CH2NH); 8.6 (m, 1H, HNCH3).
4.2.3. N,N0 -dimethylamide of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid 3 A solid residue which was purified by silica gel column chromatography (ethyl acetate: methanol, 5:1) to give 3 as a white solid. Yield 0.32 g, 89%. M.p. 96e98 C. 1H NMR (200 MHz, DMSO): d 1.45 (s, 9H, C(CH3)3); 2.82 (s, 3H, CH3N); 3.05 (s, 3H, CH3N); 3.33 (m, 2H, CHCH2NH); 3.724 (s, 3H, OCH3); 4.6 (m, 1H, CHCH2); 6.8 (ABq, 2H, CH]CH); 7.06 (m, 1H, CH2NH); 8.65 (m, 1H, C(O)NHCH).
4.3.1. Hydrochloride of N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic acid amide 4 Yield 0.55 g, 84%. M.p. 203e204 C. 1H NMR (200 MHz, D2O): d 3.74 (s, 3H, OCH3); 3.79 (d d, 2H, 3J ¼ 4.9 Hz, 2J ¼ 12.9 Hz, CHCH2NH); 4.17 (t, 1H, J ¼ 4.88 Hz, NHCHCH2); 6.8 (ABq, 2H, J ¼ 15.67 Hz, CH]CH). ESI-Q-TOF MS m/z 216.0977 [M þ H]þ
4.3.2. Hydrochloride of N3-(4-mehtoxyfumaroyl)-(S)-2,3diaminopropanoic acid N-methylamide 5 Yield 0.6 g, 87%. M.p. 190e194 C. 1H NMR (200 MHz, D2O): d 2.7 (s, 3H, NHCH3); 3.7(d d, 2H, 3J ¼ 5.8 Hz, 2J ¼ 14.1 Hz, CHCH2NH); 3.75 (s, 3H, OCH3); 4.1 (t, 1H, J ¼ 5.8 Hz NHCHCH2); 6.8 (ABq, 2H, J ¼ 15.67 Hz, CH]CH). ESI-Q-TOF MS m/z 230.1132 [M þ H]þ
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4.3.3. Hydrochloride of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid N,Ndimethylamide 6 Yield 0.150 g, 63%. M.p. 150 C. 1H NMR (200 MHz, DMSO-d6): d 2.86 (s, 3H, CH3); 3.08 (s, 3H, CH3); 3.54 (m, 2H, CHCH2NH); 3.73 (s, 3H, OCH3); 4.45 (m, 1H, NHCHCH2); 6.7 (ABq, 2H, J ¼ 15.59 Hz CH]CH); 8.35 (m, 2H, NH2CH); 9.14 (m, 1H, CH2NHC(O)). ESI-QTOF MS m/z 244,1291 [M þ H]þ 4.4. In vitro activity Minimal inhibitory concentrations (MIC's) for antifungal susceptibility were determined by the serial 2-fold dilution microtitre plate method in YNB medium with ammonium sulfate as a nitrogen source at pH 5.0 following the literature procedure [10]. Antibacterial activity was performed in Mueller Hinton Broth 2 medium (acid hydrolysate of casein, beef extract and starch at pH 7.3) in accordance with previously described method [15]. Minimal inhibitory concentration inhibiting growth of the microorganism was compared to control cultures. Microbial cell growth was measured using the microplate reader Victor3, Perkin Elmer. MIC's were defined as the lowest concentration of the inhibitor that prevent visible growth of the microorganism. Dipeptide Nva-FMDP (one of the most active FMDP peptides) was used as a reference drug. 4.5. Enzymology 4.5.1. Purification of the enzyme and determination of glucosamine-6-phosphate synthase activity C. albicans GlcN-6-P synthase cell free extract was prepared by previously described procedure [16]. The concentration of glucosamine-6-phosphate was determined according to the modified Elson-Morgan method [12]. The GlcN-6-P synthase activity was assayed using 7.5 mM D-fructose-6-phosphate, 10 mM L-glutamine, 25 mM potassium phosphate buffer (pH 6.9e7.1), inhibitor at an appropriate concentration and 1e4 mg enzyme GlcN-6-P synthase in a total volume of 400 mL incubation mixture. 4.5.2. IC50 determination The ability of compounds 4e6 to inhibit the enzyme was measured by determining a concentration of inhibitor causing 50% inhibition of the enzyme (expressed in mM). The IC50 assays were performed according to the modified procedure described previously [17]. The standard mixtures were then incubated at 37 C for 30 min. The reaction was terminated by heating at 100 C for 1 min. FMDP was used as a reference inhibitor. 4.6. Determination of the affinity to the artificial biological membrane Interactions between the tested compounds and immobilized artificial membrane were investigated using HPLC column IAM PC DD2 (Regis Technologies, Inc., Morton grove IL). The column dimensions were 3 cm 4.6 mm, particle diameter 10 mm and pore width 300 Å. The chromatographic system consisted of a UV/Vis detector Model G1315B, Vacuum degasser Model G1322A, Quaternary pump Model G1311A, (Agilent Technologies 1200 Series, Palo Alto, CA). As a mobile phase was used 0.1 M phosphate buffer (pH 7.2); the flow rate was 1 mL/min, the sample was detected at 254 nm. The dead volume of the column was determined by the retention time of citric acid t0 (50 mg/mL solution in mobile phase). The capacity factor was calculated from the retention time of tested compounds and the column void volume time t0 from the following equation: k0 IAM ¼ (tr t0)/t0 [18].
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4.7. Molecular modeling In silico all ligands, designed amides of FMDP, were built by means of the Accelrys Discovery Studio package and their geometries were optimized with the CHARMm forcefield [19]. The receptor structure used for the calculations was based on 1GMS (PDB access code) and Autodock 4.2.3 package was chosen as docking engine [20]. Since the structure of the eukaryotic enzyme is not known but all amino acids forming the glutamine binding site are completely conserved in both E. coli and C. albicans enzymes the available structure of the prokaryotic GlcN-6-P synthase was used as the receptor model. The structures of the receptor and all ligands were processed by Autodock accompanied scripts in such a way that appropriate autodock atom types and Gasteiger charges were assigned to all atoms, nonpolar hydrogens were merged with respective carbon atoms and all applicable ligands0 single bonds were marked as free to rotate. The semiflexible docking simulations were performed thus the receptor structure was rigid throughout the calculations. The docking grid box was centered at the glutamine binding site and the dimensions of the grid were set to 60 points, with default grid resolution in each direction, resulting in 22 Å gridbox length. Lamarckian genetic algorithm (LGA) was used as the search procedure with the initial population set to 150, mutation rate set to 0.02 and crossover rate to 0.8. To ensure the convergence maximum number of energy evaluations and maximum number of generations were set to 25,000,000 and 27,000 respectively. For every ligand 50 independent runs were carried out and the resulting ligand poses were clustered with the tolerance of 1.8 Å for further analysis. Acknowledgments Calculations were carried out at the Academic Computer Center sk (TASK). This work was supported by the Faculty of in Gdan sk University of Technology, Gdan sk, Poland. Chemistry, Gdan References [1] M. Nucci, J.R. Perfect, When primary antifungal therapy fails, Clin. Infect. Dis. 46 (2008) 1426e1433. € rl, Triazole antifungal agents in invasive fungal infections: a [2] C. Lass-Flo comparative review, Drugs 71 (2011) 2405e2419. [3] E. Borowski, Novel approaches in the rational design of antifungal agents of low toxicity, Il Farm. 55 (2000) 206e208. [4] J. Plumbridge, E. Vimr, Convergent pathways for utilization of the amino sugars N- acetylglucosamine, N-acetylmannosamine, and N-acetylneuraminic acid by Escherichia coli, J. Bacteriol. 181 (1999) 47e54. [5] S. Milewski, Glucosamine-6-phosphate synthase-the multi-facets enzyme, Biochim. Biophys. Acta 1597 (2002) 173e192. [6] M.G. Buse, Hexosamines, insulin resistance, and the complications of diabetes: current status, Am. J. Physiol. Endocrinol. Metab. 290 (2006) E1eE8. [7] S. Milewski, H. Chmara, R. Andruszkiewicz, E. Borowski, M. Zaremba, Antifungal peptides with novel specific inhibitors of glucosamine 6-phosphate synthase, Drugs Exp. Clin. Res. 14 (7) (1988) 461e465. [8] S. Milewski, R. Andruszkiewicz, L. Kasprzak, J. Mazerski, F. Mignini, E. Borowski, Mechanism of action of anticandidal dipeptides containing inhibitors of glucosamine-6-phosphate synthase, Antimicrob. Agents Chemother. 35 (1991) 36e43. [9] R. Andruszkiewicz, T. Zieniawa, A. Walkowiak, Anticandidal properties of Nacylpeptides containing an inhibitor of glucosamine-6-phosphate synthase, J. Enzyme Inhib. Med. Chem. 20 (2) (2005) 115e121. [10] D. Koszel, I. Ła˛ cka, K. Kozłowska-Tylingo, R. Andruszkiewicz, The synthesis and biological activity of lipophilic derivatives of bicine conjugated with N3(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP)-an inhibitor of glucosamine-6-phosphate synthase, J. Enzyme Inhib. Med. Chem. 27 (2) (2012) 167e173. [11] R. Andruszkiewicz, H. Chmara, S. Milewski, E. Borowski, Synthesis of N3fumaroyl-L-2,3-diaminopropanoic acid analogues, the irreversible inhibitors of glucosamine synthetase, Int. J. Pept. Protein Res. 27 (1986) 449e453. [12] J. Czarnecka, K. Kwiatkowska, I. Gabriel, M. Wojciechowski, S. Milewski, Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification, J. Mol. Recognit. 20 (2012) 564e570.
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[13] C. Pidgeon, S. Ong, H. Liu, X. Qiu, M. Pidgeon, et al., IAM chromatography: an in vitro screen for predicting drug membrane permeability, J. Med. Chem. 38 (1995) 590e594. [14] M.N. Isupov, G. Obmolova, S. Butterworth, M.A. Badet-Denisot, B. Badet, I. Polikarpov, J.A. Littlechild, A. Teplyakov, Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 A crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase, Structure 4 (1996) 801e810. [15] R. Andruszkiewicz, H. Chmara, S. Milewski, E. Borowski, Synthesis and biological properties of N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid dipeptides, a novel group of antimicrobial agents, J. Med. Chem. 30 (1987) 1715e1719. [16] P. Sachadyn, R. Je˛ drzejczak, S. Milewski, J. Kur, E. Borowski, Purification to
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, anticandidal activity of N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic amide derivatives e Novel inhibitors of glucosamine-6-phosphate synthase Dorota Pawlak, Magdalena Stolarska, Marek Wojciechowski, Ryszard Andruszkiewicz* sk University of Technology, Gdan sk, Poland Department of Pharmaceutical Technology and Biochemistry, Gdan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2014 Received in revised form 3 December 2014 Accepted 4 December 2014 Available online 5 December 2014
Novel FMDP amides 4e6 have been synthesized and tested against Candida strains. The anticandidal activity has been confined only to Candida albicans. Anticandidal activity of the tested amides has correlated with their inhibitory activity of glucosamine-6-phosphate synthase in cell free extract from C. albicans. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Glucosamine-6-phosphate synthase inhibitors Antifungal activity Molecular modeling FMDP analogs
1. Introduction In recent years there has been a significant increase in the number of systemic fungal infections [1]. In many cases these infections lead to a serious diseases of internal organs such as the liver or lungs. Currently used chemotherapeutics in the antifungal therapy such as azoles, only exert fungistatic activity, therefore the incidence of azole-resistant fungal strains are still increasing [2]. On the other hand the most effective antifungal antibiotic such as amphotericin B displays a number of side effects. Toxicity, resistance phenomenon, low and narrow antifungal activity of known antifungal agents prompted the search for a new chemical compounds that could be used in the treatment of fungal infections. Glucosamine-6-phosphate (GlcN-6-P) synthase, has been proposed as one of the most perspective molecular targets for antifungal and antimicrobial therapy [3]. The enzyme is involved in the synthesis pathway of the cell wall. This enzyme catalyzes the transfer of an amino group of L-glutamine to D-fructose-6-phosphate with formation of D-glucosamine-6-phosphate which is a substrate for the
* Corresponding author. Department of Pharmaceutical Technology and sk University of Technology, 11/12 Narutowicza Street, 80-233 Biochemistry, Gdan sk, Poland. Gdan E-mail address: [email protected] (R. Andruszkiewicz). http://dx.doi.org/10.1016/j.ejmech.2014.12.007 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
synthesis of macromolecules containing chitin and mannoproteins in fungi, peptidoglycan and lipopolysaccharide of bacteria, as well as glycoproteins and glycolipids in higher organisms [4,5]. Inactivation of the enzyme in microbial cells leads to inhibition of cell wall synthesis and, consequently, to lysis of the cells without negative effects for mammalian cells. Additionally, due to participation of GlcN-6-P synthase in the biosynthesis of amino sugars, the enzyme may be involved into the regulation of blood glucose levels. Therefore, specific inhibitors of GlcN-6-P synthase activity may be also regarded as new therapeutic agent for type II diabetes [6]. A large number of compounds have showed the ability to inactivate GlcN-6-P synthase and the most effective ones, have been found to be the analogs of L-glutamine, the enzyme substrate. N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (FMDP) is one of the strongest and the most specific inhibitors of GlcN-6-P synthase. The polar character of the molecule prevents its penetration into cells by free diffusion, therefore FMDP itself, showed almost no activity when tested on fungal cell. Much efforts have been devoted to improve FMDP antifungal activity. The most successful approach based on the incorporation of FMDP molecule into short peptides [7]. These peptides were transported into cells via peptide permeases, then hydrolyzed with cytoplasmic peptides with the release of FMDP inhibitor inside the
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inhibitory potency against glucosamine-6-phosphate synthase when compared to the unmodified compound FMDP. There is a correlation between the size of amide function and ability to inhibit the enzyme. The most substituted, tertiary amide of FMDP (6) displayed the highest IC50 value.
cell [8]. Although a number of FMDP peptides exerted good activity when tested in vitro a rapid formation of FMDP peptides-resistant strains of Candida albicans may hamper the future utility of these agents. In order to overcome this problem, FMDP molecule has been modified by construction of more lipophilic derivatives of FMDP, e.g. by acylating its N-terminal amino group, or esterification its carboxyl group thus increasing their transport into cell by free diffusion [9]. Latent derivatives of FMDP were also synthesized and tested [10]. Their antifungal activity however was not acceptable at the present stage of development. A series of FMDP tertiary and secondary amides with improved lipophilicity, containing four to seven carbon atoms into alkyl chains, showed however, weak inhibitory activity towards fungal GlcN-6-P synthase, and relatively poor activity against fungal species. Development of inhibitor modified at the carboxyl group of with enhanced lipophilicity may still be solution to the GlcN-6-P synthase inhibitor transport problem. Thus we have decided to explore the carboxyl group for the construction of FMDP derivatives with GlcN-6-P synthase inhibitory activity and antifungal activity. In this communication we report the design and synthesis of three novel inhibitors of GlcN-6-P synthase, i.e. the primary amide, N-methyl and N,N-dimethylamide FMDP, evaluation of anticandidal activity and inhibitory properties toward fungal GlcN-6-P synthase (Fig. 1). Since glutamine binding site of the GlcN-6-P synthase is covered by so called Q-loop and thus does not have much room for large substituents, we first investigated by molecular modeling methods, how substitution of the charged carboxyl group with neutral and bulky N-methyl and N,N-dimethylamide affects the ligand interaction with the receptor.
Data presented in Table 1 indicate that the new compounds exhibit no activity against a series of antibacterial species, including Gram-negative and Gram-positive ones. Candida glabrata and Candida parapsilosis strains seems to be insensitive to the action of novel compounds. The activity of novel compounds is only confined to the yeast of C. albicans ATCC 10231. All compounds, however, have displayed much better activity against C. albicans than FMDP itself. The results also demonstrate that anticandidal activity strongly depends on the number of substituents at nitrogen atom of amide group. Compound 4 - primary amide, has shown to be the strongest inhibitor of the fungal cells growth. The in vitro anticandidal activity of new compound is also correlated with the GlcN-6-P synthase inhibitory properties. Compound 6 displayed the lowest inhibitory activity against GlcN6-P synthase and the poorest in vitro activity against C. albicans. FMDP itself showed the best enzyme inhibitory activity and the lack of antifungal activity which can be attributed to the polar character of FMDP. It can be concluded that the higher lipophilicity of FMDP amides influenced their antifungal activity. The research of the antimicrobial and inhibition activity of the newly obtained compounds was completed by the results of molecular modeling method.
2. Results and discussion
2.4. Membrane affinity
2.1. Synthesis
The ability to diffuse through biological membrane for new derivatives and unmodified inhibitor was investigated using a HPLC column mimicking the lipidic environment found in cell membrane - IAM (immobilized artificial membrane) PC DD2 [13]. The retention times measured for the examined compounds and affinity to the biological membrane expressed as log k0 IAM values may be approximately used as a measure of their lipophilic properties (Table 2). This data present that the log k’IAM values increase with the number of methyl group at the nitrogen atom. Despite the fact that the synthesized derivatives (4e6) have rather low affinity to the membrane, their affinity however, is much better than in the case of FMDP molecule itself. The affinity to the biological membrane depends not only on the compound lipophilicity but also on number of hydrogen bonds, electrostatic interactions and sterical effects as well. Therefore, the log k0 IAM values reflects rather membrane affinity than lipophilic properties of the compound.
The synthesis (Scheme 1) started with N2-tert-butoxycarbonylN -(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid prepared by known procedure [11]. The amide, N-methylamide and N,Ndimethylamide of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diamiopropanoic acid (1e3) were obtained using mixed anhydride method in good yields as crystalline compounds. Deprotection of the Boc groups with 4 M HCl in dioxane afforded compounds 4e6 as amorphous hydrochloride salts in acceptable yields. All new compound were fully characterized by 1H NMR and mass spectrum analyses. 3
2.2. Glucosamine-6-phosphate synthase inhibition studies The new synthesized compounds as well as the parent inhibitor FMDP were tested as inhibitors of purified C. albicans GlcN-6-P synthase overproduced by Escherichia coli [12]. Their ability to inhibit this enzyme was measured by determining a concentration which caused 50% inhibition of the enzyme. The results are presented in Table 2. All the compounds (4e6) showed lower
Fig. 1. Structures of new inhibitors.
2.3. Antimicrobial activity
2.5. Molecular modeling The docking results of the primary amide derivative of FMDP to GlcN-6-P synthase glutamine binding site were very promising. The lowest energy and the most abundant ligand pose (28 results out of 50) fits into the binding pocket very well resembling the pattern of crucial interactions of the crystal ligand (HGA) (Fig. 2) [14]. Unmodified free amine group recreates the interactions of the respective group of the HGA (and native ligand e glutamine) with Gly99, Thr76 and Asp123. Despite the fact that the negatively charged carboxyl group, present in FMDP and all other inhibitors known so far, is missing, the amide moiety introduced in the designed inhibitors is still able to partially recreate the network of substrate's favorable interactions with the receptor and also form new ones. Namely the CO moiety of the amide mimics the
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Scheme 1. Syntheses of new compound (4e6). a) N-methylmorpholine, isobutyl chloroformate; b) 40% RH(aq) (R ¼ NH2,NHCH3,N(CH3)2); c) 4 M HCl/Dioxane.
interactions of the carboxyl group with sidechains of Arg73 and His86, replacing the salt bridge with strong hydrogen bonds (OeHN distances are 1.87 and 1.88 respectively). On the other hand it turns out that the NH2 moiety of the amide, that is not present neither in the crystal ligand nor in the enzyme's natural substrate, forms weak hydrogen bonds to Asp123 side chain's carboxyl group and His77 backbone CO group (the NHeO distances are 2.18 and 2.13 respectively). All these aminoacids are essential for GlcN-6-P synthase interactions with ligands and are conserved in the sequences of this enzyme from all organisms including C. albicans and E. coli [5]. Surprisingly, N-methylamide derivative of FMDP also fits well into the glutamine binding pocket of GlcN-6-P synthase, despite the bulky methyl group replacing one of the hydrogen atoms (Fig. 2). It turned out that the increased volume of the secondary amide moiety can be compensated by only small shift of the ligand while most of its essential interactions with the receptor can be preserved. The CO moiety of the secondary amide still forms the hydrogen bonds with Arg73 guanidine moiety and His86 side chain (OeHN distances 1.92 and 2.09 respectively) while remaining NH group of the amide forms weak hydrogen bonds to Asp123 sidechain's carboxyl group (the OeHN distance 2.01). Interestingly missing, due to the substitution of the second hydrogen with methyl, hydrogen bond to His77 is replaced with van der Waals interactions of the methyl group with Thr122 aliphatic part of the side chain (CeCG2 distance 3.06). Thus binding of the N-methylamide derivative of FMDP is only lowered with respect to the primary amide derivative. It is also reflected by the predicted binding free energy of the former increased by 1.5 kcal/mol. Docking of N,N-dimethylamide derivative of FMDP did not give consistent results. It seems that the tertiary amide bearing two bulky methyl groups prevents the ligand from binding in the proper orientation since there is not enough room inside the glutamine binding site. All predicted ligand poses were found to be rotated with respect to the crystal ligand and natural substrate, with N,Ndimethylamide moiety positioned outside of the binding pocket. 3. Conclusions We have synthesized three novel FMDP amides as new inhibitors of GlcN-6-P synthase. Primary amide of FMDP shows the highest anticandidal activity out of the amides tested, the activity
however was limited only to C. albicans strain. N-methyl amide of FMDP as well as its N,N-dimethylamide displayed significantly lower activity against C. albicans. The anticandidal activity of the synthesized compounds is also correlated with their inhibitory activity towards GlcN-6-P synthase. 4. Experimental 4.1. Chemistry Thin layer chromatography (TLC) was performed using Merck aluminum plates (Kieselgel 60 F254) and visualized by ultraviolet (UV) light. Column chromatography was carried out using silica gel (70e230 mesh) (Merck). MS spectrum was recorded on Agilent Technologies 6540 UDH Accurate-Mass Q-TOF. 1H NMR spectra were recorded on Gemini Varian spectrometer operating at 200 MHz using DMSO and D2O as solvents. 4.2. General procedure for the preparation of amide of N2-tertbutoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic acid 1e3 The N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic acid (FMDP) (0.95 g, 3 mmol) was dissolved in dry THF, cooled to a temperature of 5 C, then (0.33 mL, 3 mmol) N-methylmorpholine and (0.94 mL, 3 mmol) isobutyl chloroformate were added successively. After 10 min an equimolar amount of the corresponding amine solution in water [NH3 (25% aq), CH3NH2 (40% aq), NH(CH3)2 (40% aq)] was added. After 24 h of reaction, the solvent was evaporated to dryness and the residue was dissolved in ethyl acetate. The organic layer was washed with 1 M NaHSO4 solution, water, 1 M NaHCO3 solution, than the organic solvent was removed in vacuo to give the title compound as white solid. 4.2.1. Amide of N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)(S)-2,3-diaminopropanoic acid 1 Yield 0.810 g, 86%. M.p. 136e138 C. 1H NMR (200 MHz, DMSO): d 1.37 (s, 9H. C(CH3)3), 3.55 (m, 2H, CH2NH), 3.75 (s, 3H, COOCH3), 4.1 (m, 1H, CHCH2), 6.7 (d, 1H, CHNH), 6.8 (ABq, 2H, J ¼ 14.12, CH] CH), 7.25 (s, 1H, C(O)NH), 7.36 (s, 1H, C(O)NH), 8.6 (m, 1H, CH2NHCO).
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Table 1 Antimicrobial activity of compounds 4e6 expressed as MIC (mg/mL) and compared with FMDP. Co.
R]
C. albicans ATCC 10231
C. glabra ta DSM 11226
C. parapsilosis DSM S. aureus ATCC 5784 29213
S. epidermidis PCM E. coli PCM 2118 2560
B. subtilis ATCC9372
P aeruginosa ATCC 2756
4 5 6 FMDP NvaFMDP
NH2 NHCH3 N(CH3)2 e e
4 32 512 >2000 0.25
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 128
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 8
>1024 >1024 >1024 >2000 >1024
Table 2 Inhibitory data for compound 4e6 and FMDP in respect to C. albicans GlcN-6-P synthase and affinity to biological membranes. Compound
Inhibition IC50(mM)
4 5 6 FMDP
80.70 127.05 2894.01 4.03
± ± ± ±
5.90 12.57 331.74 0.26
Log k'IAM 0.362 0.193 0.118 0.812
>1024 >1024 >1024 >2000 8
4.3. General method for the preparation of hydrochloride of N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid amide 4e6 The tert-butoxycarbonyl protecting group was removed by treating of the resulting compound (1e3) with 4 M HCl in dioxane (5 mL) for 2.5 h. Then the solvent was removed in vacuo. The residue was triturated with diethyl ether and the precipitate was filtered off and dried in vacuo over KOH.
Fig. 2. Molecular modeling. Best scoring poses of the designed ligands bound to the 1gms receptor. Ligands are shown as thick sticks models, while sidechains of essential active site residues are drawn as thin sticks. Hydrogen bonds between ligands a the receptor are marked with blue dashed lines. Left e best scoring pose of the primary amide derivative of FMDP. Right e best scoring pose of the secondary amide derivative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4.2.2. N-methylamide of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid 2 Yield 0.88 g, 66%. M.p. 115 C. 1H NMR (200 MHz, DMSO): d 1.4 (s, 9H, C(CH3)3); 3.35 (s, 3H, NHCH3); 3.4 (m, 2H, CHCH2); 3.75 (s, 3H, OCH3); 4.1 (m, 1H, CHCH2); 6.8 (ABq, 2H, J ¼ 15.56, CH]CH); 6.84 (d, 1H, J ¼ 8.19, NHCH); 7.85 (m, 1H, CH2NH); 8.6 (m, 1H, HNCH3).
4.2.3. N,N0 -dimethylamide of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid 3 A solid residue which was purified by silica gel column chromatography (ethyl acetate: methanol, 5:1) to give 3 as a white solid. Yield 0.32 g, 89%. M.p. 96e98 C. 1H NMR (200 MHz, DMSO): d 1.45 (s, 9H, C(CH3)3); 2.82 (s, 3H, CH3N); 3.05 (s, 3H, CH3N); 3.33 (m, 2H, CHCH2NH); 3.724 (s, 3H, OCH3); 4.6 (m, 1H, CHCH2); 6.8 (ABq, 2H, CH]CH); 7.06 (m, 1H, CH2NH); 8.65 (m, 1H, C(O)NHCH).
4.3.1. Hydrochloride of N3-(4-methoxyfumaroyl)-(S)-2,3diaminopropanoic acid amide 4 Yield 0.55 g, 84%. M.p. 203e204 C. 1H NMR (200 MHz, D2O): d 3.74 (s, 3H, OCH3); 3.79 (d d, 2H, 3J ¼ 4.9 Hz, 2J ¼ 12.9 Hz, CHCH2NH); 4.17 (t, 1H, J ¼ 4.88 Hz, NHCHCH2); 6.8 (ABq, 2H, J ¼ 15.67 Hz, CH]CH). ESI-Q-TOF MS m/z 216.0977 [M þ H]þ
4.3.2. Hydrochloride of N3-(4-mehtoxyfumaroyl)-(S)-2,3diaminopropanoic acid N-methylamide 5 Yield 0.6 g, 87%. M.p. 190e194 C. 1H NMR (200 MHz, D2O): d 2.7 (s, 3H, NHCH3); 3.7(d d, 2H, 3J ¼ 5.8 Hz, 2J ¼ 14.1 Hz, CHCH2NH); 3.75 (s, 3H, OCH3); 4.1 (t, 1H, J ¼ 5.8 Hz NHCHCH2); 6.8 (ABq, 2H, J ¼ 15.67 Hz, CH]CH). ESI-Q-TOF MS m/z 230.1132 [M þ H]þ
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4.3.3. Hydrochloride of N2-tert-butoxycarbonyl-N3-(4methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid N,Ndimethylamide 6 Yield 0.150 g, 63%. M.p. 150 C. 1H NMR (200 MHz, DMSO-d6): d 2.86 (s, 3H, CH3); 3.08 (s, 3H, CH3); 3.54 (m, 2H, CHCH2NH); 3.73 (s, 3H, OCH3); 4.45 (m, 1H, NHCHCH2); 6.7 (ABq, 2H, J ¼ 15.59 Hz CH]CH); 8.35 (m, 2H, NH2CH); 9.14 (m, 1H, CH2NHC(O)). ESI-QTOF MS m/z 244,1291 [M þ H]þ 4.4. In vitro activity Minimal inhibitory concentrations (MIC's) for antifungal susceptibility were determined by the serial 2-fold dilution microtitre plate method in YNB medium with ammonium sulfate as a nitrogen source at pH 5.0 following the literature procedure [10]. Antibacterial activity was performed in Mueller Hinton Broth 2 medium (acid hydrolysate of casein, beef extract and starch at pH 7.3) in accordance with previously described method [15]. Minimal inhibitory concentration inhibiting growth of the microorganism was compared to control cultures. Microbial cell growth was measured using the microplate reader Victor3, Perkin Elmer. MIC's were defined as the lowest concentration of the inhibitor that prevent visible growth of the microorganism. Dipeptide Nva-FMDP (one of the most active FMDP peptides) was used as a reference drug. 4.5. Enzymology 4.5.1. Purification of the enzyme and determination of glucosamine-6-phosphate synthase activity C. albicans GlcN-6-P synthase cell free extract was prepared by previously described procedure [16]. The concentration of glucosamine-6-phosphate was determined according to the modified Elson-Morgan method [12]. The GlcN-6-P synthase activity was assayed using 7.5 mM D-fructose-6-phosphate, 10 mM L-glutamine, 25 mM potassium phosphate buffer (pH 6.9e7.1), inhibitor at an appropriate concentration and 1e4 mg enzyme GlcN-6-P synthase in a total volume of 400 mL incubation mixture. 4.5.2. IC50 determination The ability of compounds 4e6 to inhibit the enzyme was measured by determining a concentration of inhibitor causing 50% inhibition of the enzyme (expressed in mM). The IC50 assays were performed according to the modified procedure described previously [17]. The standard mixtures were then incubated at 37 C for 30 min. The reaction was terminated by heating at 100 C for 1 min. FMDP was used as a reference inhibitor. 4.6. Determination of the affinity to the artificial biological membrane Interactions between the tested compounds and immobilized artificial membrane were investigated using HPLC column IAM PC DD2 (Regis Technologies, Inc., Morton grove IL). The column dimensions were 3 cm 4.6 mm, particle diameter 10 mm and pore width 300 Å. The chromatographic system consisted of a UV/Vis detector Model G1315B, Vacuum degasser Model G1322A, Quaternary pump Model G1311A, (Agilent Technologies 1200 Series, Palo Alto, CA). As a mobile phase was used 0.1 M phosphate buffer (pH 7.2); the flow rate was 1 mL/min, the sample was detected at 254 nm. The dead volume of the column was determined by the retention time of citric acid t0 (50 mg/mL solution in mobile phase). The capacity factor was calculated from the retention time of tested compounds and the column void volume time t0 from the following equation: k0 IAM ¼ (tr t0)/t0 [18].
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4.7. Molecular modeling In silico all ligands, designed amides of FMDP, were built by means of the Accelrys Discovery Studio package and their geometries were optimized with the CHARMm forcefield [19]. The receptor structure used for the calculations was based on 1GMS (PDB access code) and Autodock 4.2.3 package was chosen as docking engine [20]. Since the structure of the eukaryotic enzyme is not known but all amino acids forming the glutamine binding site are completely conserved in both E. coli and C. albicans enzymes the available structure of the prokaryotic GlcN-6-P synthase was used as the receptor model. The structures of the receptor and all ligands were processed by Autodock accompanied scripts in such a way that appropriate autodock atom types and Gasteiger charges were assigned to all atoms, nonpolar hydrogens were merged with respective carbon atoms and all applicable ligands0 single bonds were marked as free to rotate. The semiflexible docking simulations were performed thus the receptor structure was rigid throughout the calculations. The docking grid box was centered at the glutamine binding site and the dimensions of the grid were set to 60 points, with default grid resolution in each direction, resulting in 22 Å gridbox length. Lamarckian genetic algorithm (LGA) was used as the search procedure with the initial population set to 150, mutation rate set to 0.02 and crossover rate to 0.8. To ensure the convergence maximum number of energy evaluations and maximum number of generations were set to 25,000,000 and 27,000 respectively. For every ligand 50 independent runs were carried out and the resulting ligand poses were clustered with the tolerance of 1.8 Å for further analysis. Acknowledgments Calculations were carried out at the Academic Computer Center sk (TASK). This work was supported by the Faculty of in Gdan sk University of Technology, Gdan sk, Poland. Chemistry, Gdan References [1] M. Nucci, J.R. Perfect, When primary antifungal therapy fails, Clin. Infect. Dis. 46 (2008) 1426e1433. € rl, Triazole antifungal agents in invasive fungal infections: a [2] C. Lass-Flo comparative review, Drugs 71 (2011) 2405e2419. [3] E. Borowski, Novel approaches in the rational design of antifungal agents of low toxicity, Il Farm. 55 (2000) 206e208. [4] J. Plumbridge, E. Vimr, Convergent pathways for utilization of the amino sugars N- acetylglucosamine, N-acetylmannosamine, and N-acetylneuraminic acid by Escherichia coli, J. Bacteriol. 181 (1999) 47e54. [5] S. Milewski, Glucosamine-6-phosphate synthase-the multi-facets enzyme, Biochim. Biophys. Acta 1597 (2002) 173e192. [6] M.G. Buse, Hexosamines, insulin resistance, and the complications of diabetes: current status, Am. J. Physiol. Endocrinol. Metab. 290 (2006) E1eE8. [7] S. Milewski, H. Chmara, R. Andruszkiewicz, E. Borowski, M. Zaremba, Antifungal peptides with novel specific inhibitors of glucosamine 6-phosphate synthase, Drugs Exp. Clin. Res. 14 (7) (1988) 461e465. [8] S. Milewski, R. Andruszkiewicz, L. Kasprzak, J. Mazerski, F. Mignini, E. Borowski, Mechanism of action of anticandidal dipeptides containing inhibitors of glucosamine-6-phosphate synthase, Antimicrob. Agents Chemother. 35 (1991) 36e43. [9] R. Andruszkiewicz, T. Zieniawa, A. Walkowiak, Anticandidal properties of Nacylpeptides containing an inhibitor of glucosamine-6-phosphate synthase, J. Enzyme Inhib. Med. Chem. 20 (2) (2005) 115e121. [10] D. Koszel, I. Ła˛ cka, K. Kozłowska-Tylingo, R. Andruszkiewicz, The synthesis and biological activity of lipophilic derivatives of bicine conjugated with N3(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP)-an inhibitor of glucosamine-6-phosphate synthase, J. Enzyme Inhib. Med. Chem. 27 (2) (2012) 167e173. [11] R. Andruszkiewicz, H. Chmara, S. Milewski, E. Borowski, Synthesis of N3fumaroyl-L-2,3-diaminopropanoic acid analogues, the irreversible inhibitors of glucosamine synthetase, Int. J. Pept. Protein Res. 27 (1986) 449e453. [12] J. Czarnecka, K. Kwiatkowska, I. Gabriel, M. Wojciechowski, S. Milewski, Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification, J. Mol. Recognit. 20 (2012) 564e570.
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