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      Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole

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          Abstract

          Background

          Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14α-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding.

          Methodology/Principal Findings

          We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 Å and 2.27 Å), and from the related pathogen T. brucei (resolutions of 2.7 Å and 2.6 Å), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180° depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target.

          Conclusions/Significance

          The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti-Chagasic drugs with improved efficacy and reduced toxicity.

          Author Summary

          Chagas Disease is caused by kinetoplastid protozoa Trypanosoma cruzi, whose sterols resemble those of fungi, in both composition and biosynthetic pathway. Azole inhibitors of sterol 14α-demethylase (CYP51), such as fluconazole, itraconazole, voriconazole, and posaconazole, successfully treat fungal infections in humans. Efforts have been made to translate anti-fungal azoles into a second-use application for Chagas Disease. Ravuconazole and posaconazole have been recently proposed as candidates for clinical trials with Chagas Disease patients. However, the widespread use of posaconazole for long-term treatment of chronic infections may be limited by hepatic and renal toxicity, a requirement for simultaneous intake of a fatty meal or nutritional supplement to enhance absorption, and cost. To aid our search for structurally and synthetically simple CYP51 inhibitors, we have determined the crystal structures of the CYP51 targets in T. cruzi and T. brucei, both bound to the anti-fungal drugs fluconazole or posaconazole. The structures provide a basis for a design of new drugs targeting Chagas Disease, and also make it possible to model the active site characteristics of the highly homologous Leishmania CYP51. This work provides a foundation for rational synthesis of new therapeutic agents targeting the three kinetoplastid parasites.

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          Author and article information

          Contributors
          Role: Editor
          Journal
          PLoS Negl Trop Dis
          plos
          plosntds
          PLoS Neglected Tropical Diseases
          Public Library of Science (San Francisco, USA )
          1935-2727
          1935-2735
          April 2010
          6 April 2010
          : 4
          : 4
          Affiliations
          [1 ]Department of Pharmaceutical Chemistry, University of California, San Francisco, California, United States of America
          [2 ]Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California, United States of America
          McGill University, Canada
          Author notes

          Conceived and designed the experiments: MPJ LMP. Performed the experiments: CKC SSFL LMP. Analyzed the data: CKC SSFL CG LMP. Contributed reagents/materials/analysis tools: CG MPJ JHM LMP. Wrote the paper: JHM LMP.

          Article
          09-PNTD-RA-0635R2
          10.1371/journal.pntd.0000651
          2850312
          20386598
          Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
          Page count
          Pages: 15
          Categories
          Research Article
          Biochemistry/Biomacromolecule-Ligand Interactions
          Biochemistry/Drug Discovery
          Biochemistry/Structural Genomics
          Infectious Diseases/Neglected Tropical Diseases

          Infectious disease & Microbiology

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