Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors

Busca de inibidores da enzima glicossomal gliceraldeído 3-fosfato desidrogenase de Trypanosoma cruzi

ARTIGO

Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors

Busca de inibidores da enzima glicossomal gliceraldeído 3-fosfato desidrogenase de Trypanosoma cruzi

Ana C. Leite^I; Alessandra R. P. Ambrozin^I; Marcelo S. Castilho^II; Paulo C. Vieira^I; João B. Fernandes^I,*; Glaucius Oliva^II; Maria Fátima das G. F. da Silva^I; Otávio H. Thiemann^II; M. Inês S. Lima^III; José R. Pirani^IV

^IDepartamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos-SP, Brazil

^IIInstituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos-SP, Brazil

^IIIDepartamento de Botânica, Universidade Federal de São Carlos, Caixa Postal 676, 13565-905 São Carlos-SP, Brazil

^IVDepartamento de Botânica, Instituto de Biociências, Universidade de São Paulo, 05508-900, São Paulo-SP, Brazil

ABSTRACT

The inhibitory activity of crude extracts of Meliaceae and Rutaceae plants on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) enzyme from Trypanosoma cruzi was evaluated at 100 μg/mL. Forty-six extracts were tested and fifteen of them showed significant inhibitory activity (IA % > 50). The majority of the assayed extracts of Meliaceae plants (Cedrela fissilis, Cipadessa fruticosa and Trichilia ramalhoi) showed high ability to inhibit the enzymatic activity. The fractionation of the hexane extract from branches of C. fruticosa led to the isolation of three flavonoids: flavone, 7-methoxyflavone and 3',4',5',5,7-pentamethoxyflavone. The two last compounds showed high ability to inhibit the gGAPDH activity. Therefore, the assayed Meliaceae species could be considered as a promising source of lead compounds against Chagas' disease.

Keywords: Rutaceae, Meliaceae, flavonoids, gGAPDH, Trypanosoma cruzi, inhibitors.

RESUMO

Nesse trabalho foi avaliada a atividade inibitória sobre a enzima glicossomal gliceraldeído-3-fosfato desidrogenase de T. cruzi (gGAPDH) de extratos vegetais oriundos de plantas das famílias Meliaceae e Rutaceae, na concentração de 100 μg/mL. Foram testados 46 extratos, dos quais 15 apresentaram atividade inibitória significativa (% AI > 50). A maioria dos extratos de plantas da família Meliaceae (Cedrela fissilis, Cipadessa fruticosa e Trichilia ramalhoi) apresentou grande potencial em inibir a atividade enzimática. O fracionamento do extrato hexânico dos galhos de C. fruticosa permitiu o isolamento de três flavonóides: flavona, 7-metoxiflavona e 3',4',5',5,7-pentametoxiflavona. Os dois últimos foram ativos na inibição da atividade de gGAPDH. Desta forma, as três espécies de Meliaceae testadas podem ser consideradas promissoras na busca de compostos protótipos para o controle da doença de Chagas.

Unitermo: Rutaceae, Meliaceae, flavonóides, gGAPDH, Trypanosoma cruzi, inibidores.

INTRODUCTION

Chagas' disease, caused by the protozoan Trypanosoma cruzi, affects some 16-18 million people, mostly from South and Central America, where 25 % of the total population are at risk of contamination (WHO, 2007). Control of the insect vector (Triatoma infestans) in endemic areas has led to the virtual elimination of transmission by insect bites, and as consequence, blood transfusion and congenital transmission are the major causes for the spread of the disease (Dias, 1993). Its treatment is still a challenge, since the only drug commercially available (benznidazole) has strong side effects (De Castro, 1993).

The bloodstream form of parasites of the family Trypanosomatidae possesses a microbody-like organelle, where glycolysis takes place, the glycosome (Opperdoes & Borst, 1977). The bloodstream form of the parasite T. cruzi has no functional tricarboxylic acid cycle, and it is highly dependent on glycolysis for ATP production. This great dependence on glycolysis as a source of energy makes the glycolytic enzymes attractive targets for trypanocidal drug design (Souza et al., 1998). Glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) is a glycolytic enzyme which catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate (GAP) to 1,3-bisphosphoglycerate (BGP). The three dimensional structure of T. cruzi gGAPDH previously determined shows differences, when compared to the homologous human enzyme that could be exploited for selective inhibition (Souza et al., 1998).

The need for the development of more efficient drugs against Chagas' disease has stimulated us to initiate a project aiming at the discovery of lead compounds from several plants of Rutales order. In this way, we have proposed the utilization of the glycolytic enzyme gGAPDH as a target for the search of new natural trypanocides (Tomazela et al., 2000; Vieira et al., 2001; Pavão et al., 2002; Moraes et al., 2003). In this paper, we report the gGAPDH inhibition activity of 46 crude extracts of Meliaceae and Rutaceae plants, along with three flavones isolated from an active fraction.

MATERIAL AND METHODS

Plant material

The fruits, branches, stems, roots, and leaves of Cedrela fissilis Vell. were collected in 01/06/01 in São Carlos (São Paulo, Brazil) and identified by Dr. Maria Inês Salgueiro Lima from the Department of Botany, Universidade Federal de São Carlos, where a voucher specimen (6701) was deposited. The fruits, branches and leaves of Cipadessa fruticosa Bl. were collected in 01/20/01 in Viçosa (Minas Gerais, Brazil) and identified by Dr. José R. Pirani from the Department of Botany, Universidade de São Paulo and deposited at the herbarium from this department with a voucher number 110.664. The branches of Almeidea coerulea A. St.-Hil., stems, and leaves of A. rubra A. St.-Hil., Conchocarpus heterophyllus (A. St.-Hil.) Kallunki & Pirani, Galipea carinata Pirani (sp. nov.) and Trichilia ramalhoi Rizzini were collected in Southeastern Brazil, and identified by Dr. José R. Pirani from the Department of Botany, Universidade de São Paulo, Brazil. The specimens were deposited at the Herbarium of that Department. The voucher numbers and the dates of collection were described in Ambrozin et al. (2004).

Preparation of crude extracts

The powdered air-dried plant material (branches, fruits, leaves, roots, and/or stems) was extracted by maceration three times with hexane, at room temperature for 72 h. This process was repeated with dichloromethane and methanol for C. fissilis and C. fruticosa, and only with methanol for the other species. The solvent was removed under reduced pressure by rotary evaporation and the extracts obtained were assayed on T. cruzi gGAPDH enzyme.

Isolation of compounds

The hexane extract from branches of C. fruticosa (12.2 g) was submitted to vacuum liquid chromatography over silica gel using a hexane-dichloromethane-ethyl acetate-methanol gradient. The ethyl acetate fraction (6.8 g) was chromatographed on silica gel, eluting with a hexane-dichloromethane-acetone gradient to give 9 fractions. Fraction 3 was fractionated as above, using hexane-dichloromethane-acetone (6:3:1), affording 9 fractions. Fraction 5 was three times chromatographed through column chromatography on silica gel, eluting with hexane-dichloromethane-methanol (7:2.5:0.5) to afford compounds 1 (16.7 mg) and 2 (8.2 mg). Fraction 6 was twice submitted to column chromatography with hexane-dichloromethane-methanol (6:3:1) giving compound 3 (30.6 mg). Compounds 1-3 were characterized by comparison of ¹³C NMR data with the literature (Kingsburry & Looker, 1975; Passador et al., 1997) and submitted to the enzymatic assay.

Preparation and purification of recombinant T. cruzi gGAPDH

TcGAPDH was overexpressed and purified as reported by Souza et al. (1998). It is maintained in the Crystallography Laboratory of the Universidade de São Paulo, São Carlos, SP, Brazil.

T. cruzi gGAPDH inhibitory activity

gGAPDH activity was determined according to a previously reported procedure (Barbosa & Nakano, 1987; Vieira et al., 2001). Reduced NADH was spectrophotometrically measured at 340 nm during 30 s. The reaction medium contains 50 mmol L^-1 Tris-HCl pH 8.6 buffer, 1 mmol L^-1 EDTA, 1 mmol L^-1β-mercapto-ethanol, 30 mmol L^-1 Na₂HAsO₄, 2.5 mmol L^-1 NAD⁺, 0.3 mmol L^-1 glyceraldehyde-3-phosphate and 0.15 mg protein, in a total volume of 500 μL. The reaction was initiated by the addition of substrate. The extracts were tested at a final concentration of 100 μg/mL.

RESULTS

In this work, we evaluated the inhibitory activity of 46 extracts of Meliaceae and Rutaceae plants on T. cruzi gGAPDH enzyme (Table 1). Fifteen of them showed high ability in inhibit the enzyme (IA % > 50) at 100 μg/mL. The hexane extract from branches of C. fruticosa (CFGH) and the methanol extracts from Meliaceae plants were the most active ones, whereas the extracts from Rutaceae did not present significant results.

The investigation of the ethyl acetate fraction (inhibitory activity = 81.9 % at 100 μg/mL) from the hexane extract from branches of C. fruticosa (CFGH) led to the isolation of three flavonoids: flavone (1), 7-methoxyflavone (2) and 3',4',5',5,7-pentamethoxyflavone (3). The flavones 1 and 2 were assayed (Table 2) and the results showed that only 2 presented high inhibitory activity against gGAPDH enzyme.

DISCUSSION

The results indicated that, among the assayed species, C. fissilis, C. fruticosa and Trichilia ramalhoi are promising sources of lead compounds for the rational design of new trypanocidal drugs. The inhibitory activity of the flavones 1 and 2 against gGAPDH has been reported for the first time. Recently, Ambrozin et al. (2004) published their activity against trypomastigote forms of T. cruzi and Tadesmir et al. (2006) described the trypanocidal activity of some flavones. A previous study showed that compound 3 completely inhibited the gGAPDH activity at 268.8 μmol/L (Tomazela et al., 2000). Thus, the activity of the studied extract (CFGH) (Table 1) is probably related to the presence of the active flavones 2 and 3. However, their enzymatic activity were considered weak, particularly in comparison with chalepin, a coumarin isolated from Pilocarpus spicatus (Rutaceae), which reduced the gGAPDH activity by 75 % at 93 μmol/L (IC₅₀ = 64 μmol/L) (Vieira et al., 2001; Pavão et al., 2002; Menezes et al., 2003; Moraes et al., 2003; Leitão et al., 2004).

Similar studies involving the enzyme gGAPDH have been reported by our group. Vieira et al. (2001) showed the enzymatic activity of several plants of Rutaceae and Meliaceae families and thirteen coumarins isolated from different species, which are considered as one of the most promising class of substances with gGAPDH inhibitory activity. Moreover, the gGAPDH activity of several flavonoids isolated from Meliaceae, Rutaceae and Leguminosae plants, including pyrano chalcones, flavones, flavanones, flavonols and isoflavones, have also been reported (Tomazela et al., 2000; Moraes et al., 2003). Recently, Januário et al. (2005) described the activity of 7-hydroxy-4',6-dimethoxyisoflavone on gGAPDH. All these data support the hypothesis that highly oxygenated flavones possess the structural requirements to inhibit gGAPDH.

The family Meliaceae is characterized by the frequent occurrence of limonoids (da Silva et al., 1984). This class of compounds has been attracted considerable interest because of their biological properties and diversity of structures (Champagne et al., 1992).

Previous studies of C. fissilis showed the isolation of limonoids and triterpenes (Taylor, 1984; Zelnik, 1966, 1970; Leite et al., 2005a; Ambrozin et al., 2006) and C. fruticosa has been reported to contain ent-clerodanes and labdanes diterpenoids (Rojatkar and Nagasampagi, 1994; Rojatkar et al., 1994), limonoids, steroids, sesquiterpenoids, heneicosene derivatives and one coumarin (Luo et al., 2000, 2001; Leite et al., 2005a,b). There are no phytochemical studies about T. ramalhoi, but several compounds have been isolated from the Trichilia genus, such as, limonoids (Cortez et al., 1992; Garcez et al., 1997; Garcez, et al., 2000; Cortez et al., 2000), lignan glycosides (Cortez et al., 1998), ω-phenyl alkanoic and alkenoic acids (Pupo et al., 1996), terpenoids, steroids (Pupo et al., 2002; Pupo et al., 1996) and γ-lactones (Pupo et al., 1998). Phytochemical studies with other active extracts from these plants are in progress and shall reveal molecules that can be used as potential lead compounds in the search for potent and selective T. cruzi gGAPDH inhibitors.

In conclusion, most of the active extracts belong to the family Meliaceae. New investigations with these extracts are undertaken in order to find inhibitors that could act as potential lead molecules on the described assay. Moreover, the isolated flavonoids bring about the enzymatic inhibition, showing that these compounds are potential lead molecules for the development of new drugs against Chagas' disease.

ACKNOWLEDGEMENTS

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and Financiadora de Estudos e Projetos (FINEP) for the financial support.

Received 19 August 2008

Accepted 18 December 2008

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Publication Dates

• Publication in this collection
  11 Aug 2009
• Date of issue
  Mar 2009

History

• Accepted
  18 Dec 2008
• Received
  19 Aug 2008