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Antimicrobial Agents and Chemotherapy, November 2008, p. 4098-4114, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.01616-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vitro Activities of ER-119884 and E5700, Two Potent Squalene Synthase Inhibitors, against Leishmania amazonensis: Antiproliferative, Biochemical, and Ultrastructural Effects{triangledown}

Juliany Cola Fernandes Rodrigues,1 Juan Luis Concepcion,2 Carlos Rodrigues,3 Aura Caldera,3 Julio A. Urbina,3* and Wanderley de Souza1*

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco G, subsolo, Ilha do Fundão, Rio de Janeiro, RJ 21.941-902, Brazil,1 Laboratorio de Enzimologia de Parasitos, Facultad de Ciencias, Universidad de los Andes, Merida, Venezuela,2 Laboratório de Química Biológica, Centro de Bioquímica y Biofísica, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela3

Received 17 December 2007/ Returned for modification 8 March 2008/ Accepted 20 August 2008

ER-119884 and E5700, novel arylquinuclidine derivatives developed as cholesterol-lowering agents, were potent in vitro growth inhibitors of both proliferative stages of Leishmania amazonensis, the main causative agent of cutaneous leishmaniasis in South America, with the 50% inhibitory concentrations (IC50s) being in the low-nanomolar to subnanomolar range. The compounds were very potent noncompetitive inhibitors of native L. amazonensis squalene synthase (SQS), with inhibition constants also being in the nanomolar to subnanomolar range. Growth inhibition was strictly associated with the depletion of the parasite's main endogenous sterols and the concomitant accumulation of exogenous cholesterol. Using electron microscopy, we identified the intracellular structures affected by the compounds. A large number of lipid inclusions displaying different shapes and electron densities were observed after treatment with both SQS inhibitors, and these inclusions were associated with an intense disorganization of the membrane that surrounds the cell body and flagellum, as well as the endoplasmic reticulum and the Golgi complex. Cells treated with ER-119884 but not those treated with E5700 had an altered cytoskeleton organization due to an abnormal distribution of tubulin, and many were arrested at cytokinesis. A prominent contractile vacuole and a phenotype typical of programmed cell death were frequently found in drug-treated cells. The selectivity of the drugs was demonstrated with the JC-1 mitochondrial fluorescent label and by trypan blue exclusion tests with macrophages, which showed that the IC50s against the host cells were 4 to 5 orders of magnitude greater that those against the intracellular parasites. Taken together, our results show that ER-119884 and E5700 are unusually potent and selective inhibitors of the growth of Leishmania amazonensis, probably because of their inhibitory effects on de novo sterol biosynthesis at the level of SQS, but some of our observations indicate that ER-119884 may also interfere with other cellular processes.

* Corresponding author. Mailing address for Wanderley de Souza: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco G, subsolo, Ilha do Fundão, Rio de Janeiro, RJ 21.941-902, Brazil. Phone: 55-21-25626581. Fax: 55-21-22602364. E-mail: wsouza{at}biof.ufrj.br. Mailing address for Julio A. Urbina: Laboratório de Química Biológica, Centro de Bioquímica y Biofísica, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela. Phone and fax: (513) 321-2981. E-mail: jurbina{at}mac.com

{triangledown} Published ahead of print on 2 September 2008.

Antimicrobial Agents and Chemotherapy, November 2008, p. 4098-4114, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.01616-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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