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

Abstract

BackgroundChagas 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 FindingsWe 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/SignificanceThe 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.

Sterols in spermatogenesis and sperm maturation

“…Mammalian CYP51 from different species displays over 90% sequence identity at the protein level ( 15 ). Moreover, CYP51 from distant eukaryotic organisms has been found to be strikingly similar at the structural level (16)(17)(18)(19). These data indicate that CYP51 function has remained highly conserved throughout evolution, suggesting an essential role of this gene in organisms of various taxa.…”

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

“…Our structures identify important ligand-binding constraints. The antifungal triazole drug ITC extends from the active site to just beyond the mouth of the entry channel, similar to posaconazole in the T. brucei CYP51 structure (49). The triazole group of the ITC makes a coordination bond with the heme iron.…”

Structural Insights into Binding of the Antifungal Drug Fluconazole to Saccharomyces cerevisiae Lanosterol 14α-Demethylase

“…Until 2014, X-ray structures of eukaryotic CYP51s were obtained by deletion of the transmembrane helix to improve expression and crystallization (16). These CYP51s included the human CYP51 enzyme (PDB identifiers [IDs] 3LD6, 3JUV, and 3JUS) (16) and CYP51 enzymes from the protozoa Trypanosoma cruzi and Trypanosoma brucei (PDB IDs 2WX2, 2WV2, and 2X2N) (17). Other available CYP51 structures include those of Mycobacterium tuberculosis CYP51 (e.g., PDB ID 1EA1) (19) and Leishmania infantum CYP51 (PDB ID 3L4D) (20).…”