Leishmanicidal Galloylquinic Acids are Noncompetitive Inhibitors of Arginase

Ácidos galoilquínicos, substâncias que têm apresentado atividade leishmanicida, foram isolados do extrato de acetato de etila da planta Byrsonima coccolobifolia. Estes compostos fenólicos juntamente com o ácido gálico demonstraram ser uma nova classe de inibidores não-competitivos potentes em arginase (ARG) de Leishmania amazonensis (Ki variando de 0,10 a 0,68 μmol L ). O ácido quínico não apresentou atividade inibitória significativa em ARG demonstrando que a unidade galoila tem características importantes que permitem a interação enzima-inibidor. A atividade inibitória significativa do ácido gálico frente à ARG pode ser uma indicação para o entendimento da resposta imune previamente observada em Leishmania donovani, uma vez que a atividade enzimática da arginase está associada à diminuição dos níveis de NO no processo de infecção por Leishmania.


Introduction
Leishmaniasis is a deadly infectious tropical disease caused by the protozoan of the genus Leishmania, which affects more than 12 million people of broad geographical distribution. 1,2][5] Exploring for novel therapeutic opportunities, new biochemical targets have been investigated, in particular arginase (ARG) from Leishmania amazonensis have been considered an attractive target in the search for new leishmanicidal agents.7][8] In addition, the crystal structure of ARG from Leishmania mexicana was solved, 9 making this protease more interesting to investigate for new antileishmanial compounds.
Arginase is a metalloenzyme with binuclear manganese center, which catalyzes the last step in the urea cycle in mammals, allowing hydrolyzes of L-arginine to L-ornithine and urea. 10In infected macrophages the substrate L-arginine is used by ARG and by nitric oxide synthase (NOS) in two different metabolic pathways.The reaction catalyzed by ARG is carried out in the polyamines (PAs) metabolism, essential to preserve parasite viability, and the NOS pathway generate nitric oxide (NO) molecules, which production increases oxidative stress. 11,12The balance between NOS and mammalian arginase is competitively regulated by TH 1 and TH 2 cytokines as protective response.Leishmania protozoa explore the immune response of TH 2 increasing ARG expression in host cells and consequently PAs for growth and establishment of infection. 8,13,14ol. 25, No. 10, 2014   N ω -hydroxy-L-arginine and 2(S)-amino-6boronohexanoic acid are synthetic aminoacid derivatives, which inhibit in a competitive mode ARG from L. mexicana with K i values of 85 and 1.3 µmol L -1 respectively.However, in an in vivo study these synthetic compounds could not contain the infection completely. 7,15Flavonoids isolated from plants have been reported as inhibitors of ARG from L. amazonensis.][18][19] Quinic acid is a hydrated form of shikimic acid and together with galloylquinic acids (tannic acids) and gallic acid are derivatives from shikimate pathway, available and widespread in plant sources. 20,213][24] Additionally, tannic acids have presented antileishmanial activity (EC 50 = 2-38 µmol L -1 ), and their leishmanicidal potency have been associated to the number of galloyl groups substituents in the shikimic acid moiety. 25Gallic acid singly reduces Leishmania donovani amastigotes (EC 50 = 4.4 µg mL -1 ) in murine macrophages by activating leishmanicidal macrophage functions. 26Also, tannins identified from fractions of cajazeira (Spondias mombin L.) showed leishmanicidal effect in vitro on amastigotes of Leishmania chagasi, with IC 50 in the range of 0.61 to 17.07 µg mL -1 . 27However, so far, no study has been reported on this class of secondary metabolites exploring their inhibitory activity on ARG from L. amazonensis.
Based on the usage of natural products as invaluable tools in the search for new drugs 5,28 and in the antiprotozoal activities previously found in Byrsonima species, 29,30 we performed a phytochemical study of fractions of Byrsonima coccolobifolia Kunth.(Malpighiaceae) extract that significantly inhibited ARG enzyme.][33][34][35] Galloylquinic acids (3-6) and gallic acid (2) were potent inhibitors of ARG with high affinity.Quinic acid (1) 36 isolated from Myrcia lingua (O.Berg) Mattos (Myrtaceae) was also tested on ARG to compare inhibitory activity with those galloylquinic acid derivatives, but the inhibition of ARG found for this compound was not significant.

General experimental procedures
The measurement of urea in the enzymatic assays was performed on a Beckman Coulter DU 800 spectrophotometer at 600 nm.The 1D and 2D nuclear magnetic resonance (NMR) data were acquired on a Bruker DRX-400 NMR spectrometer ( 1 H: 400 MHz; 13 C: 100 MHz) using D 2 O and MeOH-d 4 as solvents.Silica gel 60 (Merck, 230-700 mesh) and Sephadex LH-20 (Amersham Pharmacia Biotech AB) together with thin-layer chromatography (TLC) on pre-coated aluminum silica 60 F 254 (Merck) were used to isolate the compounds.Compounds were visualized in TLC UV 254/366 and by the usage of the stain sulfuric vanillin solution.The solvents ethanol (EtOH), methanol (MeOH), hexane, acetone, ethyl acetate (EtOAc) from Vetec were used to prepare the extracts and for chromatographic procedures.

Plant material
B. coccolobifolia leaves and stems were collected in the cerrado at Federal University of São Carlos (UFSCar), São Carlos -SP, Brazil.The plant material was identified by Dr Maria Inês Salgueiro Lima, and deposited at the Herbarium of the Botany Laboratory (HUFSCar) in UFSCar (voucher No. 8367).

Arginase assay
The recombinant arginase of L. amazonensis was expressed and purified as described previously. 19,36,37The kinetics measurements of ARG were performed as reported before, 10,19,39 resulting a K m value of 22.6 ± 1.7 mmol L -1 (R 2 = 0.996).Compounds were serially diluted, using at least 10 concentrations for IC 50 determinations (quinic acid (1) was diluted from 5000 to 1.22 µmol L -1 and compounds (2-6), the concentrations were between 250 and 0.024 µmol L -1 ).Mix I was prepared using 50 µL of CHES buffer solution at pH 9.6, 8 µL of arginase solution and 292 µL of water.A volume of 5 µL sample of each concentration of inhibitor was added to 35 µL of mix I, and the reaction mixture was incubated for 10 min at 37 °C.Then 10 µL of L-arginine solution was added to the reaction giving 50 mmol L -1 substrate and 50 mmol L -1 of CHES buffer at pH 9.6 in a final assay volume, which was incubated again for 10 min at 37 °C.The urea was measured spectrophotometrically at 600 nm by an enzymatic colorimetric assay, 40 using a commercially available kit (Biotécnica, Brazil).A volume of 10 µL of the enzymatic reaction was added to 500 µL of reagent 1 previously prepared (50 mmol L -1 phosphate buffer, pH 6.7, 60 mmol L -1 salicylate, 3.2 mmol L -1 sodium nitroprusside, and 30000 IU urease) and incubated at 37 °C for 10 min.After incubation, 500 µL of reagent 2 (140 mmol L -1 NaOCl and 150 mmol L -1 NaOH) was added and incubated at 37 °C for 10 min.The enzymatic assay was performed in duplicate and a negative control and a positive control (quercitrin) 16 were used.Type of inhibition of active compounds was evaluated using the same procedure, but increasing substrate concentrations in the range of 6.25-72.0mmol L -1 .K is (affinity constant of enzyme-inhibitor) and K ii (affinity constant of inhibitor with enzyme-substrate complex) for compounds (2-6) were found by double-reciprocal (Lineweaver-Burk) plots, using slopes and ordinate intercepts plotted versus the respective inhibitor concentrations in the abscissa.The constants are resulting from the straight lines (linear regression), which the abscissa intercepts leads to -K ii and -K is .2][43] In addition, Dixon plots of 1/velocity versus inhibitor concentration data was also used to find . All data analyses were carried out with the SigmaPlot 12.0 and GraFit5 software.

Results and Discussion
The quinic acid esters (3-6) and gallic acid (2), isolated from the active EtOAc fraction of B. coccolobifolia strongly Vol. 25, No. 10, 2014   inhibited ARG (Table 1).However, quinic acid (1) did not show expressive inhibition on ARG.Furthermore, high activity was demonstrated by compounds with large number of phenolic hydroxy groups.Comparing the IC 50 values for compounds (2-6) the potency of gallic acid (2) is more significant, but increasing the galloyl groups in the quinic acid moiety (3-6) did not show a considerable change in the IC 50 values, as observed previously for leishmanicidal potency in tannic acids. 25inetics studies of compounds (2-6) showed that these inhibitors shared a common behavior acting as noncompetitive inhibitors.For compounds 2, 5 and 6 the noncompetitive inhibition is also frequently known as mixed type, while inhibitors 3 and 4 are designated as pure noncompetitive inhibitors.Recently, gallic acid (K i = 7.2 ± 1.4 µmol L -1 ) and epigallocatechin-3-gallate (EGCG) (K i = 4.0 ± 0.5 µmol L -1 ) showed potent ARG inhibitory activity with a noncompetitive and mixed type, respectively. 44nalysis of Lineweaver-Burk plot (Figure 2A) for gallic acid (2) showed the intersecting lines converging to the left of the y-axis and above the abscissa (K ii > K is ), which means the inhibitor 2 binds with higher affinity to the free enzyme than to the enzyme-substrate complex.Additionally, when quinic acid is substituted in the position 5 by a galloyl (compound 3) or a methoxy galloyl moieties (compound 4), this behavior changes a little, each inhibitor (3 and 4) now binds with the same affinity to both, free enzyme and enzyme-substrate complex (K ii = K is ).This can also be seen by the double reciprocal plots (Figures 2B and 2C), which the nest of lines converge nearly at the x-axis.On the other hand, for the substituted quinic acid with two or more galloyl moieties (compound 5 and 6), the bind affinity is higher to the enzyme-substrate complex than to the free enzyme (K ii < K is ), and the line intercept is now located below the abscissa in both plots (Figures 2D and 2E).The calculated values of affinity constants (Table 1) support the findings.
Gallic acid and derivatives have been reported as responsible for benefic effects and a number of chemopreventive properties provided by green tea and wine consumption. 45,468][49][50] Additionally, some gallotannins and tannic acids have showed inhibitory potential on enzymes like α-glucosidase, fatty acid synthase (FAS) and HIV-1 protease. 24,51,524][55] The aggregate-based promiscuous inhibition could be related to the large reactive functional groups of these compounds. 55e have identified the inhibition mechanism of the quinic acid esters (3-6) and gallic acid (2) as noncompetitive and mixed inhibitors, suggesting that the inhibitory activity of ARG would not be related to the formation of aggregates.In addition, some of these phenols have already been reported as leishmanicidal compounds.Previous study showed that gallic acid increases the expression of nitric oxide synthase (iNOS) and cytokine genes in Leishmania major parasitised RAW 264.7 cells, as properties to combat the infection. 56his phenol demonstrated in in vitro study on L. donovani, a nonspecific immune response recognized by release of tumour necrosis factor (TNF) and elevated nitric oxide (NO) concentration. 26eishmania stimulate the PAs production for their development and decrease the level of NO prompted by NOS. 7,8,57Thus, ARG inhibition could turn on the production of NO, instead of PAs preventing the infection.

Conclusions
Gallic acid and derivatives isolated from B. coccolobifolia showed to be novel noncompetitive and mixed inhibitors of ARG from L. amazonensis, with K i values in the low µmol L -1 range.In conclusion, our results suggest that the ability of galloyl moiety in induce immunological response demonstrated earlier may be also a result of the inhibition of ARG activity.In this view, the galloylquinic acids should be considered as promising hits in the investigation of leishmanicidal compounds.

Table 1 .
IC 50 and K i values for secondary metabolites active on ARG aQuercitrin is a noncompetitive inhibitor used as a positive control (K ii and K is obtained from the reference 18); b data values represent means of individual experiments ± SD.