Oxidosqualene Cyclase Inhibitors as Antimicrobial Agents Jerald C. Hinshaw, ‡ Dae-Yeon Suh, ‡ Philippe Garnier, ‡ Frederick S. Buckner, § Richard T. Eastman, § Seiichi P. T. Matsuda, ⊥ Bridget M. Joubert, ⊥ Isabelle Coppens, | Keith A. Joiner, | Salim Merali, † Theodore E. Nash, # and Glenn D. Prestwich* ,‡ Department of Medicinal Chemistry, University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah 84108-1257, Department of Medicine, University of Washington, Box 357185, Seattle, Washington, 98195, Department of Chemistry and Department of Biochemistry and Cell Biology, Rice University, 6100 South Main Street, Houston, Texas 77005, Infectious Disease Section, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022, Department of Medical and Molecular Parasitology, New York University School of Medicine, 341 East 25th Street, New York, New York 10010, and Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received June 9, 2003 Abstract: Small-molecule oxidosqualene cyclase (OSC) in- hibitors were found to be effective in assays against cloned OSC-like enzymes from human pathogens. A combinatorial library was prepared and used to identify lead compounds that inhibit the growth of Trypanosoma cruzi, Leishmania mexi- cana amazonensis, and Pneumocystis carinii in culture. Se- lectivity for the microorganisms in preference to mammalian cells was observed. Introduction. The world’s population continues to be seriously plagued by pathogenic organisms. In third world countries, millions are afflicted with diseases such as leishmaniasis and trypanosomiasis. Even in more developed societies, immunosuppression from organ transplants, cancer chemotherapy, and the AIDS epi- demic has resulted in the increased appearance of grave opportunistic infections, such as Pneumocystis pneu- monia and toxoplasmosis. New treatments for all these pathogens are desperately needed. Current drugs for these illnesses show toxicity, limited effectiveness, or pathogen resistance, which leads to considerable suf- fering and millions of deaths worldwide annually. Sterol and triterpene biosynthetic pathways are wide- spread in living organisms because these polycyclic compounds are vital in cell membrane function and, in some cases, are hormonal effectors. 1,2 As a result, inhibitors of terpene biosynthesis have found successful applications as antifungals, herbicides, and drugs, 1 including the notable statin family of HMG-CoA reduc- tase inhibitors, for controlling cholesterol levels in humans. 3-5 Oxidosqualene cyclases (OSCs) are impor- tant enzymes in triterpenoid biosynthesis. 6 These en- zymes catalyze the cyclization of (3S)-2,3-oxidosqualene to lanosterol in fungi, mammals, and some protists and to cycloartenol, as well as several other pentacyclic triterpenes in plants 1 (Scheme 1). Recent studies have revealed that a number of human pathogenic microorganisms synthesize sterols. These include Pneumocystis carinii (pneumonia), 7 Trypano- soma brucei (African sleeping sickness), 8 Trypanosoma cruzi (Chagas disease), 9 and Leishmania sp. (leish- maniasis). 10-12 Sterol biosynthetic routes have been examined as drug targets against Trypanosome 13,14 and Pneumocystis 15 organisms. Because hypercholesterolemia is a major risk factor for the development of atherosclerosis in humans, considerable research and development have been di- rected toward the inhibition of pathways in cholesterol biosynthesis. OSC is a pivotal enzyme in the biosyn- thesis of cholesterol in humans. 1 A potential approach toward cholesterol level modulation would be the selec- tive inhibition of OSC. Several small-molecule OSC inhibitors have been designed and synthesized for this purpose and are in various stages of preclinical and clinical evaluation. 2,16-22 Exploiting this ongoing development effort, we envis- aged that OSCs present in human pathogens might be inhibited by known OSC inhibitors, thus providing new leads to antibiotics and antiparasitic agents. Inhibition of the key sterol cyclization step in pathogens is attrac- tive because it is downstream in the overall pathway. Therefore, microbes using either the mevalonate or methylerythritol phosphate pathways for isoprenoid product biosynthesis 23-25 would be targeted. Results and Discussion. To test this hypothesis, we evaluated two known OSC inhibitors 16,22 (Figure 1) against several organisms in cell assays. For our initial experiments, we examined human pathogenic microor- ganisms from three groups. The first group included organisms known or suspected of utilizing OSC-like enzymes (Pneumocystis sp., Leishmania sp., Trypano- soma sp.). 7,9,13 In the second group was Toxoplasma gondii, which is now believed to rely minimally, if at all, on de novo sterol synthesis. 26,27 Giardia lamblia, 28 known not to biosynthesize sterols, was examined as a representative of group three. In these experiments, Chinese hamster ovary (CHO) cells provided a bench- mark for effects on mammalian cells. Activities were noted to be consistent with what is known about sterol synthesis in these organisms. As shown in Figure 2, microorganisms with known sterol synthesis pathways are affected by the two OSC inhibitors with varying sensitivities. Thus, T. cruzi was more sensitive to II, while P. carinii was more sensitive to I. These two OSC * To whom correspondence should be addressed. Phone: 801-585- 9051. Fax: 801-585-9053. E-mail: glenn.prestwich@hsc.utah.edu. ‡ University of Utah. § University of Washington. ⊥ Rice University. | Yale University. † New York University. # National Institutes of Health. Scheme 1. Cyclization of Oxidosqualene 4240 J. Med. Chem. 2003, 46, 4240-4243 10.1021/jm034126t CCC: $25.00 © 2003 American Chemical Society Published on Web 08/29/2003