Targeted Genome Mining Reveals the Biosynthetic Gene Clusters of Natural Product CYP51 Inhibitors.

Lanosterol 14α-demethylase (CYP51) is an important target in the development of antifungal drugs. The fungal-derived restricticin 1 and related molecules are the only examples of natural products that inhibit CYP51. Here, using colocalizations of genes encoding self-resistant CYP51 as the query, we identified and validated the biosynthetic gene cluster (BGC) of 1. Additional genome mining of related BGCs with CYP51 led to production of the related lanomycin 2. The pathways for both 1 and 2 were identified from fungi not known to produce these compounds, highlighting the promise of the self-resistance enzyme (SRE) guided approach to bioactive natural product discovery.

Lead optimization generates selenium-containing miconazole CYP51 inhibitors with improved pharmacological profile for the treatment of fungal infections.

A series of selenium-containing miconazole derivatives were identified as potent antifungal drugs in our previous study. Representative compound A03 (MIC = 0.01 µg/mL against C.alb. 5314) proved efficacious in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03 exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-containing miconazole derivatives resulted in the discovery of similarly potent compound B17 (MIC = 0.02 µg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity against Candida albicans and significant effects on the treatment of resistant Candida albicans infections. Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising in vivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In addition, molecular docking studies provide a model for the interaction between the compound B17 and the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be further developed for the potential treatment of fungal infections.

Novel mannopyranoside esters as sterol 14α-demethylase inhibitors: Synthesis, PASS predication, molecular docking, and pharmacokinetic studies.

Direct unimolar one-step valeroylation of methyl α-d-mannopyranoside (MDM) furnished mainly 6-O-valeroate. However, similar reaction catalyzed by DMAP resulted 3,6-di-O-valeroate (21%) and 6-O-valeroate (47%) indicating reactivity sequence as 6-OH>3-OH>2-OH,4-OH. To get potential antimicrobial agents, 6-O-valeroate was converted into four 2,3,4-di-O-acyl esters, and 3,6-di-O-valeroate was converted into 2,4-di-O-acetate. Direct tetra-O-valeroylation of MDM gave a mixture of 2,3,4,6-tetra-O-valeroate and 2,3,6-tri-O-valeroate indicating that the C2-OH is more reactive than the equatorial C4-OH. The activity spectra analysis along with in vitro antimicrobial evaluation clearly indicated that these novel MDM esters had better antifungal activities over antibacterial agents. In this connection, molecular docking indicated that these MDM esters acted as competitive inhibitors of sterol 14α-demethylase (CYP51), an essential enzyme for clinical target to cure several infectious diseases. Furthermore, pharmacokinetic studies revealed that these MDM esters may be worth considering as potent candidates for oral and topical administration. Structure activity relationship (SAR) affirmed that saturated valeric chain (C5) in combination with caprylic (C8) chains was more promising CYP51 inhibitor over conventional antifungal antibiotics.

Discovery of Novel Fungal Lanosterol 14α-Demethylase (CYP51)/Histone Deacetylase Dual Inhibitors to Treat Azole-Resistant Candidiasis.

Invasive fungal infections (particularly candidiasis) are emerging as severe infectious diseases worldwide. Because of serious antifungal drug resistance, therapeutic efficacy of the current treatment for candidiasis is limited and associated with high mortality. However, it is highly challenging to develop novel strategies and effective therapeutic agents to combat drug resistance. Herein, the first generation of lanosterol 14α-demethylase (CYP51)-histone deacetylase (HDAC) dual inhibitors was designed, which exhibited potent antifungal activity against azole-resistant clinical isolates. In particular, compounds 12h and 15j were highly active both in vitro and in vivo to treat azole-resistant candidiasis. Antifungal mechanism studies revealed that they acted by blocking ergosterol biosynthesis and HDAC catalytic activity in fungus, suppressing the function of efflux pump, yeast-to-hypha morphological transition, and biofilm formation. Therefore, CYP51-HDAC dual inhibitors represent a promising strategy to develop novel antifungal agents against azole-resistant candidiasis.

Pfcyp51 exclusively determines reduced sensitivity to 14α-demethylase inhibitor fungicides in the banana black Sigatoka pathogen Pseudocercospora fijiensis.

The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Sterol 14α-Demethylase Structure-Based Optimization of Drug Candidates for Human Infections with the Protozoan Trypanosomatidae.

Sterol 14α-demethylases (CYP51) are cytochrome P450 enzymes essential for sterol biosynthesis in eukaryotes and therapeutic targets for antifungal azoles. Multiple attempts to repurpose antifungals for treatment of human infections with protozoa (Trypanosomatidae) have been undertaken, yet so far none of them have revealed sufficient efficacy. VNI and its derivative VFV are two potent experimental inhibitors of Trypanosomatidae CYP51, effective in vivo against Chagas disease, visceral leishmaniasis, and sleeping sickness and currently under consideration as antiprotozoal drug candidates. However, VNI is less potent against Leishmania and drug-resistant strains of Trypanosoma cruzi and VFV, while displaying a broader spectrum of antiprotozoal activity, and is metabolically less stable. In this work we have designed, synthesized, and characterized a set of close analogues and identified two new compounds (7 and 9) that exceed VNI/VFV in their spectra of antiprotozoal activity, microsomal stability, and pharmacokinetics (tissue distribution in particular) and, like VNI/VFV, reveal no acute toxicity.

Synthesis and fungicidal activity of novel imidazole-based ketene dithioacetals.

Novel imidazole-based ketene dithioacetals show impressive in planta activity against the economically important plant pathogens Alternaria solani, Botryotinia fuckeliana, Erysiphe necator and Zymoseptoria tritici. Especially derivatives of the topical antifungal lanoconazole, which bear an alkynyloxy or a heteroaryl group in the para-position of the phenyl ring, exhibit excellent control of the mentioned phytopathogens. These compounds inhibit 14α -demethylase in the sterol biosynthesis pathway of the fungi. Synthesis routes starting from either benzaldehydes or acetophenones as well as structure-activity relationships are discussed in detail.

Alkylated Piperazines and Piperazine-Azole Hybrids as Antifungal Agents.

The extensive use of fluconazole (FLC) and other azole drugs has caused the emergence and rise of azole-resistant fungi. The fungistatic nature of FLC in combination with toxicity concerns have resulted in an increased demand for new azole antifungal agents. Herein, we report the synthesis and antifungal activity of novel alkylated piperazines and alkylated piperazine-azole hybrids, their time-kill studies, their hemolytic activity against murine erythrocytes, as well as their cytotoxicity against mammalian cells. Many of these molecules exhibited broad-spectrum activity against all tested fungal strains, with excellent minimum inhibitory concentration (MIC) values against non-albicans Candida and Aspergillus strains. The most promising compounds were found to be less hemolytic than the FDA-approved antifungal agent voriconazole (VOR). Finally, we demonstrate that the synthetic alkylated piperazine-azole hybrids do not function by fungal membrane disruption, but instead by disruption of the ergosterol biosynthetic pathway via inhibition of the 14α-demethylase enzyme present in fungal cells.

Stepwise design, synthesis, and in vitro antifungal screening of (Z)-substituted-propenoic acid derivatives with potent broad-spectrum antifungal activity.

Fungal infections are a main reason for the high mortality rate worldwide. It is a challenge to design selective antifungal agents with broad-spectrum activity. Lanosterol 14α-demethylase is an attractive target in the design of antifungal agents. Seven compounds were selected from a number of designed compounds using a rational docking study. These compounds were synthesized and evaluated for their antifungal activity. In silico study results showed the high binding affinity to lanosterol 14α-demethylase (-24.49 and -25.83 kcal/mol) for compounds V and VII, respectively; these values were greater than those for miconazole (-18.19 kcal/mol) and fluconazole (-16.08 kcal/mol). Compound V emerged as the most potent antifungal agent among all compounds with a half maximal inhibitory concentration of 7.01, 7.59, 7.25, 31.6, and 41.6 µg/mL against Candida albicans, Candida parapsilosis, Aspergillus niger, Trichophyton rubrum, and Trichophyton mentagrophytes, respectively. The antifungal activity for most of the synthesized compounds was more potent than that of miconazole and fluconazole.

Structural insight into the unique binding properties of pyridylethanol(phenylethyl)amine inhibitor in human CYP51.

Sterol 14α-demethylase (CYP51) is the main drug target for the treatment of fungal infections. The discovery of new efficient fungal CYP51 inhibitors requires an understanding of the structural requirements for selectivity for the fungal over the human ortholog. In this study, a binding mode of the pyridylethanol(phenylethyl)amine type CYP51 inhibitor to the human ortholog was determined at the atomic level. We isolated and purified a full-length human CYP51. The inhibitor-specific binding and its conformational and dynamic properties were evaluated using UV-visible and NMR spectroscopy. Considering the experimental data in docking calculations and molecular dynamics simulations, the location of the inhibitor moieties and their interactions with the enzyme active site were determined. The inhibitor binds to the enzyme in two diastereomeric forms, which have a common location of aromatic ring moieties, while the less bulky propyl chain can adapt to various hydrophobic regions of the enzyme active site. The halogenated phenyl ring binds in the substrate access channel making numerous contacts with the hydrophobic side chains, and its interactions with the unconserved residues are especially informative. The results reveal the unique binding properties of the investigated inhibitor in comparison to the azoles and provide novel directions for the design of selective fungal inhibitors.

Shape and pharmacophore-based virtual screening to identify potential cytochrome P450 sterol 14α-demethylase inhibitors.

Sterol 14α-demethylase (CYP51) is a cytochrome P450 heme thiolate containing enzyme involved in biosynthesis of membrane sterols, including sterol in animals, ergosterol in fungi, and a variety of C24-modified sterols in plants and protozoa. Several clinical drugs have been developed to reduce the impact of fungal diseases, but their clinical uses have been limited by the emergence of drug resistance and insufficiencies in their antifungal activity. Therefore, in order to identify potential CYP51 inhibitors, we have implemented a virtual screening (VS) protocol by using both phase shape and pharmacophore model (AHHRR) against Asinex, ChemBridge and Maybridge databases. A filtering protocol, including Lipinski filter, number of rotatable bonds and different precisions of molecular docking was applied in hits selection. The results indicated that both shape-based and pharmacophore-based screening yielded the best result with potential inhibitors. The searched compounds were also evaluated with ADME properties, which show excellent pharmacokinetic properties under the acceptable range. We identified potential CYP51 inhibitors for further investigation, they could also be employed to design ligands with enhanced inhibitory potencies and to predict the potencies of analogs to guide synthesis/or prepare synthetic antifungal analogs against CYP51.

Antitrypanosomal lead discovery: identification of a ligand-efficient inhibitor of Trypanosoma cruzi CYP51 and parasite growth.

Chagas disease is caused by the intracellular protozoan parasite Trypanosomal cruzi , and current drugs are lacking in terms of desired safety and efficacy profiles. Following on a recently reported high-throughput screening campaign, we have explored initial structure-activity relationships around a class of imidazole-based compounds. This profiling has uncovered compounds 4c (NEU321) and 4j (NEU704), which are potent against in vitro cultures of T. cruzi and are greater than 160-fold selective over host cells. We report in vitro drug metabolism and properties profiling of 4c and show that this chemotype inhibits the T. cruzi CYP51 enzyme, an observation confirmed by X-ray crystallographic analysis. We compare the binding orientation of 4c to that of other, previously reported inhibitors. We show that 4c displays a significantly better ligand efficiency and a shorter synthetic route over previously disclosed CYP51 inhibitors, and should therefore be considered a promising lead compound for further optimization.

[SPR-biosensor assay for analysis of small compounds interaction with human cytochrome P450 51A1 (CYP51A1)].

The SPR assay for human cytochrome P450 51A1's (CYP51A1) ligand screening was developed. Assay has been validated with known azole inhibitors of cytochrome P450s. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR assay was showed with 19 steroid and triterpene compounds, which were not investigated as potential ligands of CYP51A1.

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Delta7-24, 25-dihydrolanosterol.

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.

Molecular modeling of human lanosterol 14α-demethylase complexes with substrates and their derivatives.

Lanosterol 14α-demethylase (CYP51A1) is a key enzyme in sterol biosynthesis. In humans, this enzyme is involved in the cholesterol biosynthesis pathway. The majority of antifungal drugs are aimed at the inhibition of CYP51 in fungi. To elucidate the molecular mechanisms of highly specific protein-ligand recognition, we have developed a full-atomic model of human CYP51A1 and performed docking of natural substrates and their derivatives to the active site of the enzyme. The parameters of the binding enthalpy of substrates, intermediates, and final products of the reaction of 14α-demethylation were estimated using the MMPB(GB)SA algorithm. Dynamic properties and conformational changes of the protein globule upon binding of the ligand near the active site have been investigated by the molecular dynamics method. Our studies reveal that hydroxylated intermediate reaction products have a greater affinity than the initial substrates, which facilitates the multistage reaction without accumulation of intermediate products. The contribution to the free energy of steroid ligand binding of 30 amino acids forming the substrate-binding region of CYP51A1, as well as the influence of their substitutions to alanine on the stability of the protein molecule, has been clarified using alanine scanning modeling. We demonstrate that the most serious weakening of the binding is observed in the case of substitutions Y137A, F145A, V149A, I383A, and R388A. The results of molecular modeling are in agreement with the data obtained by analysis of primary sequences of representatives of the CYP51 family.

In vivo investigation in pigs of intestinal absorption, hepatobiliary disposition, and metabolism of the 5α-reductase inhibitor finasteride and the effects of coadministered ketoconazole.

The overall aim of this detailed investigation of the pharmacokinetics (PK) and metabolism of finasteride in pigs was to improve understanding of in vivo PK for this drug and its metabolites. Specific aims were to examine the effects of ketoconazole coadministration on the PK in three plasma compartments (the portal, hepatic, and femoral veins), bile, and urine and to use these data to study in detail the intestinal absorption and the liver extraction ratio and apply a semiphysiological based PK model to the data. The pigs received an intrajejunal dose of finasteride (0.8 mg/kg) either alone (n = 5) or together with ketoconazole (10 mg/kg) (n = 5) or an intravenous dose (0.2 mg/kg) (n = 3). Plasma, bile, and urine (collected from 0 to 6 h) were analyzed with ultraperformance liquid chromatography-tandem mass spectrometry. Ketoconazole increased the bioavailability of finasteride from 0.36 ± 0.23 to 0.91 ± 0.1 (p < 0.05) and the terminal half-life from 1.6 ± 0.4 to 4.0 ± 1.1 h (p < 0.05). From deconvolution, it was found that the absorption rate from the intestine to the portal vein was rapid, and the product of the fraction absorbed and the fraction that escaped gut wall metabolism was high (f(a) · F(G) ∼ 1). Interestingly, the apparent absorption rate constant (k(a)) to the femoral vein was lower than that to the portal vein, probably because of binding and distribution within the liver. The liver extraction ratio was time-dependent and varied with the two routes of administration. After intrajejunal administration, from 1 to 6 h, the liver extraction ratio was significantly (p < 0.05) reduced by ketoconazole treatment from intermediate (0.41 ± 0.21) to low (0.21 ± 0.10).

Comparison of the inhibitory profiles of itraconazole and cimetidine in cytochrome P450 3A4 genetic variants.

CYP3A4, an important drug-metabolizing enzyme, is known to have genetic variants. We have previously reported that CYP3A4 variants such as CYP3A4.2, 7, 16, and 18 show different enzymatic kinetics from CYP3A4.1 (wild type). In this study, we quantitatively investigated the inhibition kinetics of two typical inhibitors, itraconazole (ITCZ) and cimetidine (CMD), on CYP3A4 variants and evaluated whether the genetic variation leads to interindividual differences in the extent of CYP3A4-mediated drug interactions. The inhibitory profiles of ITCZ and CMD on the metabolism of testosterone (TST) were analyzed by using recombinant CYP3A4 variants. The genetic variation of CYP3A4 significantly affected the inhibition profiles of the two inhibitors. In CYP3A4.7, the K(i) value for ITCZ was 2.4-fold higher than that for the wild-type enzyme, whereas the K(i) value for CMD was 0.64-fold lower. In CYP3A4.16, the K(i) value for ITCZ was 0.54-fold lower than that for wild-type CYP3A4, whereas the K(i) value for CMD was 3.2-fold higher. The influence of other genetic variations also differed between the two inhibitors. Docking simulations could explain the changes in the K(i) values, based on the accessibility of TST and inhibitors to the heme moiety of the CYP3A4 molecule. In conclusion, the inhibitory effects of an inhibitor differ among CYP3A4 variants, suggesting that the genetic variation of CYP3A4 may contribute, at least in part, to interindividual differences in drug interactions mediated by CYP3A4 inhibition, and the pattern of the influences of genetic variation differs among inhibitors as well as substrates.

A sterol 14α-demethylase is required for conidiation, virulence and for mediating sensitivity to sterol demethylation inhibitors by the rice blast fungus Magnaporthe oryzae.

The Magnaporthe oryzae genome contains two homologous CYP51 genes, MoCYP51A and MoCYP51B, that putatively encode sterol 14α-demethylase enzymes. Targeted gene deletion mutants of MoCYP51A were morphologically indistinguishable from the isogenic wild type M. oryzae strain Guy11 in vegetative culture, but were impaired in both conidiation and virulence. Deletion of MoCYP51B did not result in any obvious phenotypic changes compared with Guy11. The Δmocyp51A mutants were also highly sensitive to sterol demethylation inhibitor (DMI) fungicides, while Δmocyp51B mutants were unchanged in their sensitivity to these fungicides. Expression of both MoCYP51A and MoCYP51B was significantly induced by exposure to DMI fungicides. Analysis of intracellular localization of MoCyp51A showed that MoCyp51A was mainly localized to the cytoplasm of hyphae and conidia. Taken together, our results indicate that MoCYP51A is required for efficient conidiogenesis, full virulence and for mediating DMI sensitivity by the rice blast fungus.