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Structural basis for rational design of inhibitors targeting Trypanosoma cruzi sterol 14α-demethylase: two regions of the enzyme molecule potentiate its inhibition.
Friggeri L, Hargrove TY, Rachakonda G, Williams AD, Wawrzak Z, Di Santo R, De Vita D, Waterman MR, Tortorella S, Villalta F, Lepesheva GI
(2014) J Med Chem 57: 6704-17
MeSH Terms: 14-alpha Demethylase Inhibitors, Carbamates, Crystallography, X-Ray, Drug Design, Imidazoles, Models, Molecular, Protein Binding, Protein Conformation, Stereoisomerism, Sterol 14-Demethylase, Trypanocidal Agents, Trypanosoma cruzi
Show Abstract · Added February 12, 2015
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.
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12 MeSH Terms
In vitro and in vivo studies of the antiparasitic activity of sterol 14α-demethylase (CYP51) inhibitor VNI against drug-resistant strains of Trypanosoma cruzi.
Soeiro Mde N, de Souza EM, da Silva CF, Batista Dda G, Batista MM, Pavão BP, Araújo JS, Aiub CA, da Silva PB, Lionel J, Britto C, Kim K, Sulikowski G, Hargrove TY, Waterman MR, Lepesheva GI
(2013) Antimicrob Agents Chemother 57: 4151-63
MeSH Terms: 14-alpha Demethylase Inhibitors, Animals, Chagas Disease, Drug Resistance, Endoplasmic Reticulum, Golgi Apparatus, Imidazoles, Male, Mice, Microscopy, Electron, Transmission, Nitroimidazoles, Oxadiazoles, Protozoan Proteins, Sterol 14-Demethylase, Thiazoles, Triazoles, Trypanocidal Agents, Trypanosoma cruzi
Show Abstract · Added March 7, 2014
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.
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18 MeSH Terms
Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Delta7-24, 25-dihydrolanosterol.
Hargrove TY, Wawrzak Z, Liu J, Waterman MR, Nes WD, Lepesheva GI
(2012) J Lipid Res 53: 311-20
MeSH Terms: 14-alpha Demethylase Inhibitors, Binding Sites, Crystallography, X-Ray, Dose-Response Relationship, Drug, Enzyme Activation, Lanosterol, Models, Molecular, Mutation, Protein Conformation, Sterol 14-Demethylase, Trypanocidal Agents, Trypanosoma brucei brucei
Show Abstract · Added February 12, 2015
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.
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12 MeSH Terms
Targeting Trypanosoma cruzi sterol 14α-demethylase (CYP51).
Lepesheva GI, Villalta F, Waterman MR
(2011) Adv Parasitol 75: 65-87
MeSH Terms: 14-alpha Demethylase Inhibitors, Antifungal Agents, Azoles, Catalytic Domain, Chagas Disease, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Drug Discovery, Humans, Protein Conformation, Substrate Specificity, Triazoles, Trypanocidal Agents, Trypanosoma cruzi
Show Abstract · Added February 12, 2015
There are at least two obvious features that must be considered upon targeting specific metabolic pathways/enzymes for drug development: the pathway must be essential and the enzyme must allow the design of pharmacologically useful inhibitors. Here, we describe Trypanosoma cruzi sterol 14α-demethylase as a promising target for anti-Chagasic chemotherapy. The use of anti-fungal azoles, which block sterol biosynthesis and therefore membrane formation in fungi, against the protozoan parasite has turned out to be highly successful: a broad spectrum anti-fungal drug, the triazole compound posaconazole, is now entering phase II clinical trials for treatment of Chagas disease. This review summarizes comparative information on anti-fungal azoles and novel inhibitory scaffolds selective for Trypanosomatidae sterol 14α-demethylase through the lens of recent structure/functional characterization of the target enzyme. We believe our studies open wide opportunities for rational design of novel, pathogen-specific and therefore more potent and efficient anti-trypanosomal drugs.
Copyright © 2011 Elsevier Ltd. All rights reserved.
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14 MeSH Terms
Podophyllotoxin analogues active versus Trypanosoma brucei.
Uddin MJ, Smithson DC, Brown KM, Crews BC, Connelly M, Zhu F, Marnett LJ, Guy RK
(2010) Bioorg Med Chem Lett 20: 1787-91
MeSH Terms: Anti-Inflammatory Agents, Non-Steroidal, Cell Line, Humans, Indomethacin, Podophyllotoxin, Trypanocidal Agents, Trypanosoma brucei brucei
Show Abstract · Added March 7, 2014
In an effort to discover novel anti-trypanosomal compounds, a series of podophyllotoxin analogues coupled to non-steroidal anti-inflammatory drugs (NSAIDs) has been synthesized and evaluated for activity versus Trypanosoma brucei and a panel of human cell lines, revealing compounds with low nano-molar potencies. It was discovered that coupling of NSAIDs to podophyllotoxin increased the potencies of both compounds over 1300-fold. The compounds were shown to be cytostatic in nature and seem to act via de-polymerization of tubulin in a manner consistent with the known activities of podophyllotoxin. The potencies against T. brucei correlated directly with LogP values of the compounds, suggesting that the conjugates are acting as hydrophobic tags allowing podophyllotoxin to enter the cell.
Copyright 2010 Elsevier Ltd. All rights reserved.
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7 MeSH Terms
Crystal structures of Trypanosoma brucei sterol 14alpha-demethylase and implications for selective treatment of human infections.
Lepesheva GI, Park HW, Hargrove TY, Vanhollebeke B, Wawrzak Z, Harp JM, Sundaramoorthy M, Nes WD, Pays E, Chaudhuri M, Villalta F, Waterman MR
(2010) J Biol Chem 285: 1773-80
MeSH Terms: Amino Acid Sequence, Benzamides, Biocatalysis, Crystallography, X-Ray, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Drug Design, Enzyme Inhibitors, Humans, Ligands, Microsomes, Models, Molecular, Protein Conformation, Sterol 14-Demethylase, Sterols, Substrate Specificity, Trypanocidal Agents, Trypanosoma brucei brucei, Trypanosomiasis, African
Show Abstract · Added March 7, 2014
Sterol 14alpha-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 A resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.
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19 MeSH Terms