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Sterol 14α-demethylases (CYP51) are the cytochrome P450 enzymes required for biosynthesis of sterols in eukaryotes, the major targets for antifungal agents and prospective targets for treatment of protozoan infections. Human CYP51 could be and, for a while, was considered as a potential target for cholesterol-lowering drugs (the role that is now played by statins, which are also in clinical trials for cancer) but revealed high intrinsic resistance to inhibition. While microbial CYP51 enzymes are often inhibited stoichiometrically and functionally irreversibly, no strong inhibitors have been identified for human CYP51. In this study, we used comparative structure/functional analysis of CYP51 orthologs from different biological kingdoms and employed site-directed mutagenesis to elucidate the molecular basis for the resistance of the human enzyme to inhibition and also designed, synthesized, and characterized new compounds. Two of them inhibit human CYP51 functionally irreversibly with their potency approaching the potencies of azole drugs currently used to inhibit microbial CYP51.
With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14α-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: ()-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against and , pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Rapidly multiplying cancer cells synthesize greater amounts of cholesterol to build their membranes. Cholesterol-lowering drugs (statins) are currently in clinical trials for anticancer chemotherapy. However, given at higher doses, statins cause serious side effects by inhibiting the formation of other biologically important molecules derived from mevalonate. Sterol 14α-demethylase (CYP51), which acts 10 steps downstream, is potentially a more specific drug target because this portion of the pathway is fully committed to cholesterol production. However, screening a variety of commercial and experimental inhibitors of microbial CYP51 orthologs revealed that most of them (including all clinical antifungals) weakly inhibit human CYP51 activity, even if they display high apparent spectral binding affinity. Only one relatively potent compound, (R)-N-(1-(3,4'-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VFV), was identified. VFV has been further tested in cellular experiments and found to decrease proliferation of different cancer cell types. The crystal structures of human CYP51-VFV complexes (2.0 and 2.5 Å) both display a 2:1 inhibitor/enzyme stoichiometry, provide molecular insights regarding a broader substrate profile, faster catalysis, and weaker susceptibility of human CYP51 to inhibition, and outline directions for the development of more potent inhibitors.
Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.
Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14α-demethylase (CYP51) inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic potencies of a new set of β-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC50=1.2 nM, vs (S)-2/(S)-3, EC50=1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the development of more effective antiparasitic drugs.
Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-piperazin-1-yl)-2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α-demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azole-bound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51-targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens.
Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 μM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.
Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.
Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.
Leishmaniasis is a major health problem that affects populations of ∼90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14α-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14α-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V(max) of ∼10 and 8 min(-1), respectively), it is also found to 14α-demethylate C4-dimethylated lanosterol (V(max) = 0.9 min(-1)) and C4-desmethylated 14α-methylzymosterol (V(max) = 1.9 min(-1)). Binding parameters with six sterols were tested, with K(d) values ranging from 0.25 to 1.4 μM. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14α-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.
Pathogenic protozoa threaten lives of several hundred million people throughout the world and are responsible for large numbers of deaths globally. The parasites are transmitted to humans by insect vectors, more than a hundred of infected mammalian species forming reservoir. With human migrations, HIV-coinfections, and blood bank contamination the diseases are now spreading beyond the endemic tropical countries, being found in all parts of the world including the USA, Canada and Europe. In spite of the widely appreciated magnitude of this health problem, current treatment for sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi) and leishmaniasis (Leishmania spp.) remains unsatisfactory. The drugs are decades old, their efficacy and safety profiles are unacceptable. This review describes sterol 14α-demethylase, an essential enzyme in sterol biosynthesis in eukaryotes and clinical target for antifungal azoles, as a promising target for antiprotozoan chemotherapy. While several antifungal azoles have been proven active against Trypanosomatidae and are under consideration as antiprotozoan agents, crystal structures of sterol 14α-demethylases from three protozoan pathogens, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum provide the basis for the development of new, highly potent and pathogen-specific drugs with rationally optimized pharmacological properties.