Squalene synthase as a chemotherapeutic target in Trypanosoma cruzi and Leishmania mexicana Julio A. Urbina a, , Juan Luis Concepcion b , Salome ´ Rangel b , Gonzalo Visbal c , Renee Lira a a Laboratorio de Quimica Biolo ´gica, Centro de Bioquimica y Bioﬁsica, Instituto Venezolano de Investigaciones Cientiﬁcas, Caracas 1020, Venezuela b Laboratorio de Sı ´ntesis Orga ´nica, Centro de Quimica, Instituto Venezolano de Investigaciones Cientiﬁcas, Caracas 1020, Venezuela c Laboratorio de Enzimologı ´a de Para ´sitos, Departamento de Biologia, Facultad de Ciencias, Universidad de los Andes, Merida, Venezuela Received 18 July 2002; received in revised form 16 August 2002; accepted 16 August 2002 Abstract Trypanosoma cruzi and Leishmania parasites have a strict requirement for specific endogenous sterols (ergosterol and analogs) for survival and growth and cannot use the abundant supply of cholesterol present in their mammalian hosts. Squalene synthase (SQS, E.C. 2.5.1.21) catalyzes the first committed step in sterol biosynthesis and is currently under intense study as a possible target for cholesterol-lowering agents in humans, but it has not been investigated as a target for anti-parasitic chemotherapy. SQS is a membrane-bound enzyme in both T. cruzi epimastigotes and Leishmania mexicana promastigotes with a dual subcellular localization, being almost evenly distributed between glycosomes and mitochondrial/microsomal vesicles. Kinetic studies showed that the parasite enzymes display normal Michaelis  /Menten kinetics and the values of the kinetic constants are comparable to those of the mammalian enzyme. We synthesized and purified 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH), a potent and specific inhibitor of mammalian SQS and found that it is also a powerful non-competitive inhibitor of T. cruzi and L. mexicana SQS, with K i ’s in the range of 12  /62 nM. BPQ-OH induced a dose-dependent reduction of proliferation the extracellular stages of these parasites with minimal growth inhibitory concentrations (MIC) of 10 /30 mM. Growth inhibition and cell lysis induced by BPQ-OH in both parasites was associated with complete depletion of endogenous squalene and sterols, consistent with a blockade of de novo sterol synthesis at the level of SQS. BPQ-OH was able to eradicate intracellular T. cruzi amastigotes from Vero cells cultured at 37 8C, with a MIC of 30 mM with no deleterious effects on host cells. Taken together, these results support the notion that SQS inhibitors could be developed as selective anti-trypanosomatid agents. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Parasitic protozoa; Squalene synthase; Enzyme kinetics; Sterol biosynthesis inhibitors; Chemotherapy; Ergosterol; Episterol; 5-Dihydro- episterol 1. Introduction Trypanosoma cruzi and Leishmania parasites are responsible for large parasitic disease loads and socio- economic losses, particularly in developing counties [1  / 3]. Specific chemotherapy of the diseases caused by these protozoa remains unsatisfactory as currently available drugs have limited activity, frequent toxic side effects and rising drug resistance [1,4 /7]. However, these parasites have a strict requirement for specific endogen- ous sterols (ergosterol and analogs) for survival and growth and cannot use the abundant supply of choles- terol present in their mammalian host [6 /8]. Ergosterol biosynthesis inhibitors with potent in vitro activity and special pharmacokinetic properties in mammals (large volumes of distribution and long half-lives) can induce radical parasitological cure in animal models of both acute and chronic experimental Chagas disease and are also active against several forms of leishmaniasis [6  /9].  Corresponding author. Tel.:  /58-212-504-1660; fax:  /58-212- 504-1093 E-mail address: jaurbina@ivic.ve (J.A. Urbina). Molecular & Biochemical Parasitology 125 (2002) 35  /45 www.parasitology-online.com 0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0166-6851(02)00206-2