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Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole.

ABSTRACT: 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 14alpha-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.We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 A and 2.27 A), and from the related pathogen T. brucei (resolutions of 2.7 A and 2.6 A), 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 degrees 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.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.

SUBMITTER: Chen CK

PROVIDER: S-EPMC2850312 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole.

Chen Chiung-Kuang CK Leung Siegfried S F SS Guilbert Christophe C Jacobson Matthew P MP McKerrow James H JH Podust Larissa M LM

PLoS neglected tropical diseases 20100406 4

<h4>Background</h4>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 ...[more]

PMID: 20386598

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Project description:BackgroundChagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority.Methodology/principal findingsThe similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14?-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ?104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM.Conclusions/significanceThe hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ?-hydroxylases (CYP4), 17?-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.

Project description:Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (p<0.05), suggesting cytokinesis dysfunction. The survival of CYP51RNAi Doxycycline-treated mice (p = 0.053) and of CYP51RNAi 5-day pre-induced Doxycycline-treated mice (p = 0.008) were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC50) were 8.5, 2.7, 1.6 and 0.12 μM for T. b. brucei 427 90-13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893) and T. b. gambiense Biyamina (MHOM/SD/82), respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.

Dataset Information

Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole.

Publications

Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole.

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