Azole Inhibitors Research Articles

Identification of novel and potent triazoles targeting CYP51 for antifungal: Design, synthesis, and biological study

Invasive fungal infections (IFIs) are emerging as a serious infectious disease worldwide. Due to the lack of effective antifungal drugs and serious drug resistance, the number of people with low immunity is increasing, leading to high morbidity and mortality. Azole drugs targeting CYP51 are widely used in the treatment of invasive fungal infections. By analyzing representative azole antifungal drugs, the characteristics of pharmacophore were summarized. The binding mode of lead compound Iodiconazole was analyzed, and it was found that the narrow hydrophobic cavity was not fully occupied. Therefore, a series of triazole compounds were designed and synthesized by fragment growth strategy. Most of the compounds showed strong inhibitory activity against pathogenic fungi, among which compound A33 showed excellent inhibitory activity against pathogenic fungi and drug-resistant strains. In addition, the preferred compound A33 can prevent fungal phase transition, the formation of fungal biofilm, and show satisfactory fungicidal activity. In addition, the compound A33 was almost non-toxic to mammalian HUVEC cell. These results strongly suggested that compound A33 was worth further investigation as a potential azole inhibitor.

Kinetic insight of multi-molecule adsorption behaviors into the anticorrosion performance of azole inhibitors: A reactive force field molecular dynamics study

Density functional theory (DFT) calculations and molecular dynamics (MD) simulations are established as powerful tools in understanding the anti-corrosion mechanism of inhibitors at the atomic scale. Despite their widespread popularity, DFT calculations typically focus on small adsorption models while the inhibition efficiency of inhibitors is determined by multi-molecule adsorption film on metal surface. Classical MD simulations neglect the formation of chemical bonding, which frequently governs the overall adsorption process. In this contribution, reactive force field (ReaxFF) MD simulations were employed to overcome these limitations and investigate the differences in inhibitory capabilities among three classic azole compounds: imidazole (IDZ), 1,2,4-triazole (TAZ), and benzotriazole (BTA) on copper. Electrochemical tests and microscopic observations revealed that BTA demonstrated superior inhibitory effect on copper corrosion compared to IDZ and TAZ. However, the widely used descriptor, adsorption energy (Eads) in DFT calculations for predicting the inhibition ability of inhibitors were very similar, failing to explain the enormous variation in inhibition effectiveness. To explain this disparity, multi-molecule adsorption kinetics based on ReaxFF simulations were conducted. The results showed that BTA consistently demonstrated faster and more frequent adsorption on copper surface compared to the other two. The significant variance in inhibitory efficiency among the three inhibitors was elucidated for the first time from the kinetic insight.

Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein-Ligand Interactions of CYP51 of Candida krusei Azole-Resistant Fungal Strain.

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.

The structure and characterization of human cytochrome P450 8B1 supports future drug design for nonalcoholic fatty liver disease and diabetes

Human cytochrome P450 8B1 (CYP8B1) is involved in conversion of cholesterol to bile acids. It hydroxylates the steroid ring at C12 to ultimately produce the bile acid cholic acid. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. Herein, the human CYP8B1 protein was generated and used to identify and characterize interactions with a series of azole inhibitors, which tend to be poorly selective P450 inhibitors. Structurally related miconazole, econazole, and tioconazole bound with submicromolar dissociation constants and were effective inhibitors of the native reaction. CYP8B was cocrystallized with S-tioconazole to yield the first X-ray structure. This inhibitor bound in the active site with its azole nitrogen coordinating the heme iron, consistent with inhibitor binding and inhibition assay data. Additionally, the CYP8B1 active site was compared with similar P450 enzymes to identify features that may facilitate the design of more selective inhibitors. Selective inhibitors should promote a better understanding of the role of CYP8B1 inhibition in normal physiology and disease states and provide a possible treatment for nonalcoholic fatty liver disease and type 2 diabetes.

Endogenous Sterol Synthesis Is Dispensable for Trypanosoma cruzi Epimastigote Growth but Not Stress Tolerance.

In addition to scavenging exogenous cholesterol, the parasitic kinetoplastid Trypanosoma cruzi can endogenously synthesize sterols. Similar to fungal species, T. cruzi synthesizes ergostane type sterols and is sensitive to a class of azole inhibitors of ergosterol biosynthesis that target the enzyme lanosterol 14α-demethylase (CYP51). In the related kinetoplastid parasite Leishmania donovani, CYP51 is essential, yet in Leishmania major, the cognate enzyme is dispensable for growth; but not heat resistance. The essentiality of CYP51 and the specific role of ergostane-type sterol products in T. cruzi has not been established. To better understand the importance of this pathway, we have disrupted the CYP51 gene in T. cruzi epimastigotes (ΔCYP51). Disruption of CYP51 leads to accumulation of 14-methylated sterols and a concurrent absence of the final sterol product ergosterol. While ΔCYP51 epimastigotes have slowed proliferation compared to wild type parasites, the enzyme is not required for growth; however, ΔCYP51 epimastigotes exhibit sensitivity to elevated temperature, an elevated mitochondrial membrane potential and fail to establish growth as intracellular amastigotes in vitro. Further genetic disruption of squalene epoxidase (ΔSQLE) results in the absence of all endogenous sterols and sterol auxotrophy, yet failed to rescue tolerance to stress in ΔCYP51 parasites, suggesting the loss of ergosterol and not accumulation of 14-methylated sterols modulates stress tolerance.

Design, synthesis, and evaluation of novel 3-thiophene derivatives as potent fungistatic and fungicidal reagents based on a conformational restriction strategy

Invasive fungal infections (IFIs) are emerging as serious infectious diseases worldwide, and due to the lack of effective antifungal agents and serious drug resistance, the limited efficacy of existing drugs has led to high morbidity and mortality in patients. We optimized the lead compound 7 by conformational restriction strategy to obtain a series of 3-thiophene phenyl compounds, of which compound 21b showed excellent inhibitory activity against pathogenic and drug-resistant fungi. In addition, the preferred compound 21b could prevent the formation of fungal biofilms and displayed satisfactory fungicidal activity. Furthermore, compound 21b was almost non-toxic to mammalian THLE2 and RAW264.7 cells and did not pose a risk of drug-drug interactions. These results strongly suggested that compound 21b is worthy of further study as a potential azole inhibitor.

Impact of Pharmacogenetics on Intravenous Tacrolimus Exposure and Conversions to Oral Therapy

CYP3A5 and CYP3A4 are the predominant enzymes responsible for tacrolimus metabolism; however only a proportion of the population expresses CYP3A5 secondary to genetic variation. CYP3A5 is expressed in both the intestine and the liver and has been shown to impact both the bioavailability and metabolism of orally administered tacrolimus. Increasing the initial tacrolimus dose by 50% to 100% is recommended in patients who are known CYP3A5 expressers; however, whether this dose adjustment is appropriate for i.v. tacrolimus administration is unclear. The objective of this study was to evaluate the impact of CYP3A5 genotype as well as other pharmacogenes on i.v. tacrolimus exposure to determine whether the current genotype-guided dosing recommendations are appropriate for this formulation. In addition, this study aimed to investigate dose conversion requirements among CYP3A5 genotypes when converting from i.v. to p.o. tacrolimus. This study is a retrospective chart review of all patients who underwent allogeneic stem cell transplantation at Michigan Medicine between June 1, 2014, and March 1, 2018, who received i.v. tacrolimus at the time of their transplantation. Secondary use samples were obtained for genotyping CYP3A5, CYP3A4, and ABCB1. Patient demographic information, tacrolimus dosing and trough levels, and concomitant medications received at the time of tacrolimus trough were collected retrospectively from the patients' medical records. The i.v. dose-controlled concentration (C/D) and the i.v.:p.o. exposure ratio was calculated for all tacrolimus doses and patients, respectively. The impact of CYP3A5, CYP3A4, and ABCB1 genotypes on the i.v. C/D were evaluated with linear mixed modeling. The impact of CYP3A5 genotype on the i.v.:p.o. ratio was evaluated while controlling for age and concomitant use of an azole inhibitor. CYP3A5 and CYP3A4 genotypes were significantly associated with the i.v. C/D, with CYP3A5 expressers and CYP3A4 rapid metabolizers having 20% lower tacrolimus exposure. Neither genotype remained significant in the multivariable model, although age, hematocrit, and concomitant use of strong azole inhibitors were associated with increased i.v. C/D. When controlling for patient age and sex, CYP3A5 expressers had significantly higher i.v.:p.o. ratios than CYP3A5 nonexpressers (3.42 versus 2.78; P=.04). Post hoc analysis showed that the i.v.:p.o. ratio may differ among different CYP3A5 genotypes and azole inhibitor combinations. This study demonstrates that the current genotype-guided tacrolimus dose adjustment recommendations are inappropriate for CYP3A5 expressers receiving i.v. tacrolimus. Although CYP3A5 genotype is likely a minor contributor to i.v. tacrolimus exposure, genotype, in addition to capturing concomitant CYP3A inhibitors, would likely improve i.v.:p.o. dose conversion selection. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Effect of Benzotriazole and 5-Methyl/1-H Carboxyl Benzotriazole on Chemical Mechanical Polishing of Cobalt in H2O2 Based Slurry

In this study, the passivation mechanism of three azole inhibitors with different functional groups, benzotriazole (BTA), 5-methyl-benzotriazole (TTA), 1-H carboxyl benzotriazole (CBT), on cobalt in H2O2 based slurry were investigated. Results showed that cobalt (Co) has the highest removal rate (RR) and static etching rate (SER) in the solution without inhibitors, and RR and SER of Co decreased when three inhibitors were added to the reference solution respectively. However, compared with BTA and TTA, CBT showed the strongest passivation effect. Through the single frequency EIS experiments and X-ray photoelectron spectroscopy (XPS) measurements analysis, all three azole inhibitors can react with Co2+ to form insoluble networked nanoparticles on the Co surface, but CBT had the thickest passivation film, which was inferred that the carboxyl group on the CBT can not only increase the coverage area on the Co surface like the methyl group, but also the oxygen on the carboxyl group can chemically adsorb on the Co surface like the N17 on the imidazole ring. Moreover, TTA showed better passivation effect than BTA due to the existence of the methyl on TTA, which can increase the coverage area on Co surface and prevent the polishing slurry from contacting Co surface.

Anti-biofilm effect by the combined action of fluconazole and acetylsalicylic acid against species of Candida parapsilosis complex

The Candida parapsilosis complex has been associated with highly refractory infections mainly due to the presence of biofilms. High glucose levels enable the development of this virulence factor which can aggravate the clinical condition of patients with diabetes mellitus, those using parenteral nutrition, with invasive medical device, including others. Combined antifungal therapy, such as azole and cyclooxygenase inhibitors, may be an alternative in such infections since they modulate prostaglandin production favoring the adhesion and development of biofilms. Thus, the present study aimed to evaluate the influence of glucose supplementation in the formation and detection of Candida parapsilosis complex biofilms and to treat them using fluconazole and a cyclooxygenase inhibitor in combination. Protein spectra evaluation allowed the differentiation between species from the complex (score > 2) in our studies. All isolates were able to form active biofilms at different glucose concentrations. In addition, a significant reduction in biofilm formation was observed when fluconazole and acetylsalicylic acid were combined. The ultrastructural analysis presented typical biofilm characteristics by species from the complex. These data support new combined therapies for the treatment of fungal infections, especially with those which are resistant and therapeutic failure is associated with virulence factors.

A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis

The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.

Corrosion control of copper wiring by barrier CMP slurry containing azole inhibitor: Combination of simulation and experiment

In the chemical mechanical polishing (CMP) process of barrier layer of multilayer copper wiring in integrated circuits, it is necessary to add appropriate corrosion inhibitor into the polishing slurry to protect copper from corrosion, reduce the height difference with the barrier material and achieve high planarization. There are few literatures about the behavior of inhibitors on metal surface in CMP studied by computational chemistry. In this study, density functional calculation and Monte Carlo simulation combined with electrochemical and CMP experiments were used to investigate the adsorption and corrosion inhibition of azole inhibitors (BTA, TAZ and TT-LYK) on copper wiring in barrier layer in glycine/hydrogen peroxide reference solution system. The results show that all the azole inhibitors in the reference solution system have obvious corrosion inhibition on copper surface, and the inhibition effect of azole inhibitors was positively correlated with the increase of concentration. In particular, TT-LYK has a stronger corrosion inhibition effect, which was verified by electrochemical and CMP measurements, MD results support there are more adsorption sites between TT-LYK and copper surface. In addition, the stability of the solution containing TT-LYK and the state of copper surface evaluated by SEM after TT-LYK treatment are better. The combination of molecular dynamics methods and experiments approach may well be able to study corrosion inhibitors at the micro level and foretell better molecular structures which is more suitable to improve the efficiency of wiring corrosion inhibition.

Mechanistic Insights into Cytochrome P450 Inactivation by Azole Drugs in Acanthamoeba castellanii

Cytochrome P450 (CYPs) are heme containing enzymes that oxidize substances using iron and are capable to metabolize wide variety of xenobiotics and play key role in the activation or inactivation of various therapeutic agents. Studies have described more than 50,000 CYPs in most forms of life i.e. archaea, viruses, protists, bacteria, animals, plants and fungi.One of the major advancements in pharmacology is the discovery of antifungal activity of azole compounds. These compounds showed a very significant advancement in the management of fungal infections. Major enzyme inhibited by these azoles is 14 alpha‐demethylase, a cytochrome P450 enzyme, which is involved in the conversion of lanosterol to ergosterol.As these CYPs descend from a common ancestor and share structural features, we tried to repurpose these antifungal drugs against Acanthamoeba castellanii, an opportunistic pathogen and an emerging cause of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis.In the present study, we structurally characterized recombinant CYP51 from Acanthamoeba castellanii (AcCYP51), which is also an important enzyme in the biosynthesis of ergosterol. Interestingly, structure‐based analysis of ligand free and ligand bound forms of AcCYP51 made very remarkable observations. Here, we observed that ligand free AcCYP51 tends to dimerize both in solution (as observed in size exclusion chromatography,SEC) and in the crystals (1.8 Å) via N‐terminal swapping forming a large homo‐dimerization interface exceeding 2000 Å2. Spectral analysis of dimeric and small monomeric fractions of AcCYP51, which we obtained after SEC, have shown that only dimeric form of AcCYP51 has spectral characteristics typical of functionally competent CYP enzymes. This is for the first time that a strong and functionally competent CYP dimer was defined structurally. Further structural analysis of inhibitor (azole) bound AcCYP51 have shown that binding of inhibitors promoted dimer dissociation and we obtained AcCYP51‐inhibitor complexes in a monomeric form (2.9 Å). We have used here different azole inhibitors (according to their potency against Acanthamoeba ) and observed that according to their in‐vivo potency, these inhibitors elucidated differences in their mode of action. With a less potent inhibitor AcCYP51 was capable to restore the monomer structure whereas with the most potent inhibitor AcCYP51failed to fully restore the monomer structure. This failure of CYP51 to fully refold upon dimer dissociation, in vivo may cause an irreversible loss of a structurally aberrant target enzyme through proteolytic degradation. This proposed inactivation mechanism is consistent with the superior potency of this molecule against A. castellaniicells.Repurposing of the antifungal azole drugs (known as conazoles) emerges as a promising strategy to combat Acanthamoeba infections. The drug‐target crystal structures determined in this work and the mechanistic insights thus gained may be further exploited for the medicinal chemistry efforts aimed at improving potency and bioavailability of these drugs for applications in human diseases caused by infection with Acanthamoeba.

Targeting Orthosteric and Allosteric Pockets of Aromatase via Dual-Mode Novel Azole Inhibitors.

Breast cancer (BC) is the most diffused cancer type in women and the second leading cause of death among the female population. Effective strategies to fight estrogen responsive (ER+) BC, which represents 70% of all BC cases, rely on estrogen deprivation, via the inhibition of the aromatase enzyme, or the modulation of its cognate estrogen receptor. Current clinical therapies significantly increased patient survival time. Nevertheless, the onset of resistance in metastatic BC patients undergoing prolonged treatments is becoming a current clinical challenge, urgently demanding to devise innovative strategies. In this context, here we designed, synthesized, and performed in vitro inhibitory tests on the aromatase enzyme and distinct ER+/ER- BC cell line types of novel azole bridged xanthones. These compounds are active in the low μM range and behave as dual-mode inhibitors, targeting both the orthosteric and the allosteric sites of the enzyme placed along one access channel. Classical and quantum-classical molecular dynamics simulations of the new compounds, as compared with selected steroidal and nonsteroidal inhibitors, provide a rationale to the observed inhibitory potency and supply the guidelines to boost the activity of inhibitors able to exploit coordination to iron and occupation of the access channel to modulate estrogen production.

New Triazole and Isoxazole Compounds as Corrosion Inhibitors for Cu-Ni (90/10) Alloy and Galvanized Steel Substrates

Three organic compounds containing azole rings 1-benzyl-4-phenyl-1H-1,2,3-triazole (I1), 5-phenyl-3-propylisoxazole (I2), and 3,5-diphenylisoxazole (I3) were synthesized and characterized as corrosion inhibitors on galvanized steel and copper-nickel (Cu-Ni, 90/10) substrates. The three tested azole inhibitors acted as corrosion inhibitors on galvanized steel in the presence of real cooling water used as electrolyte. Electrochemical performance indicated that the presence of heteroatoms, such as N and O, with free pair electrons, the chain length, and the aromatic ring plays an important role in the capacity to inhibit corrosion of galvanized steel. 3,5-diphenylisoxazole (I3) has the best inhibition activity with jcorr of 2.04 × 10−7 (5 ppm), 7.57 × 10−8 (10 ppm), and 1.73 × 10−8 (20 ppm) A cm−2, followed by compounds I1 and I2. The synthesized compounds are comparable or even higher than the electrochemical performance of commercial inhibitor tolyltriazole (TTA, jcorr = 3.06 × 10−8 A cm−2). Conversely, for Cu-Ni (90/10) substrates, commercial TTA with 20 ppm displayed the lowest corrosion current densities (jcorr = 3.20 × 10−8 A cm−2) through a characteristic anodic pseudo-passivation; however, pits were observed after ~550 mVAg/AgCl. In contrast, the synthesized compounds tended to suppress anodic copper dissolution at positive potentials but, in the best case, displayed greater corrosion rates I3 (1.463 mils per year (MPY)), I2 (1.515 MPY), and I1 (0.677 MPY) in comparison with TTA (0.091 MPY). The low inhibition efficiency (IE) of synthesized compounds is correlated to the weak absorption on the copper surface.

The characterisation of two members of the cytochrome P450 CYP150 family: CYP150A5 and CYP150A6 from Mycobacterium marinum

BackgroundActinobacteria, including the Mycobacteria, have a large component of cytochrome P450 family monooxygenases. This includes Mycobacterium tuberculosis, M. ulcerans and M. marinum, and M. vanbaalenii. These enzymes can abstract CH bonds and have important roles in natural product biosynthesis. MethodsTwo members of the bacterial CYP150 family, CYP150A5 and CYP150A6 from M. marinum, were produced, purified and characterised. The potential substrate ranges of both enzymes were analysed and the monooxygenase activity of CYP150A5 was reconstituted using a physiological electron transfer partner system. CYP150A6 was structurally characterised by X-ray crystallography. ResultsCYP150A5 was shown to bind various norisoprenoids and terpenoids. It could regioselectively hydroxylate β-ionol. The X-ray crystal structure of substrate-free CYP150A6 was solved to 1.5 Å. This displayed an open conformation with short F and G helices, an unresolved F-G loop region and exposed active site pocket. The active site residues could be identified and important variations were found among the CYP150A enzymes. Haem-binding azole inhibitors were identified for both enzymes. ConclusionsThe structure of CYP150A6 will facilitate the identification of physiological substrates and the design of better inhibitors for members of this P450 family. Based on the observed differences in substrate binding preference and sequence variations among the active site residues, their roles are predicted to be different. General significanceMultiple CYP150 family members were found in many bacteria and are prevalent in the Mycobacteria including several human pathogens. Inhibition and structural data are reported here for these enzymes for the first time.

Can the inhibition of cytochrome P450 in aquatic invertebrates due to azole fungicides be estimated with in silico and in vitro models and extrapolated between species?

Azole fungicides, designed to halt fungal growth by specific inhibition of fungal cytochrome P450 (CYP51), inhibit cytochrome P450s involved in the metabolism of xenobiotics in several non-target organisms thus raising environmental concern. The present study investigates the degree by which inhibition strengths of azoles toward cytochrome P450 in rat liver, the insect Chironomus riparius larvae and the snail Lymnaea stagnalis can be extrapolated from estimated in silico affinities. Azoles’ affinities toward human cytochrome P450 isoforms involved in xenobiotic metabolism (CYP3A4, CYP2C9 and CYP2D6) as well as fungal CYP51 were estimated with a ligand-protein docking model based on the ChemScore scoring function. Estimated affinities toward the selected enzymatic structures correlated strongly with measured inhibition strengths in rat liver (ChemScore vs. logIC50 among cytochrome P450 isoforms: −0.662 < r < −0.891, n = 17 azoles), while weaker correlations were found for C. riparius larvae (−0.167 < r < −0.733, n = 9) and L. stagnalis (−0.084 < r < −0.648, n = 8). Inhibition strengths toward C. riparius and rat liver activities were found to be highly correlated to each other (r: 0.857) while no significant relationship was found between either of the species and L. stagnalis. The inhibition of cytochrome P450 due to azole fungicides could be estimated in vitro and to a lesser extent in silico for C. riparius but not for L. stagnalis, possibly due to different enzymatic susceptibility toward azole inhibition among the species.

Effects of miconazole/clotrimazole and praziquantel combinations against the liver fluke Opisthorchis felineus in vivo and in vitro.

The liver fluke Opisthorchis felineus (Rivolta, 1884) is the causative agent of opisthorchiasis felinea in Eurasia. Opisthorchiasis is a serious human and fish-eating animal's disease affecting bile ducts and the gall bladder. Currently, the main drug for specific therapy of opisthorchiasis is praziquantel. We have previously shown that azole inhibitors of O. felineus cytochrome P450 significantly reduced survival of the worms in vitro. Here, we studied in vitro anthelmintic effects of drug combinations involving azole substances approved by the US Food and Drug Administration together with praziquantel against adult or juvenile O. felineus liver flukes. A synergistic interaction was shown for praziquantel-clotrimazole (CI = 0.68) combination and for praziquantel-miconazole (CI = 0.68) combination against adult helminths in vitro. Praziquantel-miconazole (CI = 0.30) had a strongly synergistic effect against newly excysted metacercariae. We also tested anthelmintic effects of azole substances and their combinations with praziquantel in vivo in an animal model of chemotherapy. The treatment of juvenile worms (1day postinfection) with 100mg/kg miconazole resulted in a worm burden reduction (WBR) of 37.5% (P = 0.049), with 100mg/kg clotrimazole causing a WBR of 31.25% (P = 0.025). The treatment of adult worms (5-6weeks postinfection) with 100mg/kg or 200mg/kg miconazole yielded a WBR of 23.8% (P = 0.01) and 21.4% (P = 0.006), respectively. When praziquantel was administered together with clotrimazole or with miconazole, a WBR slightly greater than the effect of ED50 praziquantel was observed (WBR of 59.5 and 54.7%, respectively).In conclusion, the synergistic effect of the praziquantel-clotrimazole and praziquantel-miconazole combinations observed in vitro was not confirmed in vivo. Thus, this combination chemotherapy revealed no benefits over praziquantel monotherapy in the treatment of opisthorchiasis felinea.

Molecular dynamic modeling of CYP51B in complex with azole inhibitors

Cytochrome P450 14α-sterol demethylase (CYP51), the key enzyme in sterol biosynthesis pathway, is an important target protein of cholesterol-lowering agents, antifungal drugs, and herbicides. CYP51B enzyme is one of the CYP51 family members. In the present study, we have chosen four representative inhibitors of CYP51B: diniconazole (Din), fluconazole (Flu), tebuconazole (Teb), and voriconazole (Vor), and launched to investigate the binding features of CYP51B-inhibitor and gating characteristics via molecular docking and molecular dynamics (MD) simulations. The results show that the ranking of binding affinities among these inhibitors is Din > Teb > Vor > Flu. Din shows the strongest binding affinity, whereas Flu shows the weakest binding affinity. More importantly, based on the calculated binding free energy results, we hypothesize that the nonpolar interactions are the most important contributors, and three key residues (Thr77, Ala258, and Lys454) play crucial role in protein-inhibitor binding. Besides, residue Phe180 may play a molecular switch role in the process of the CYP51B-Teb and CYP51B-Vor binding. Additionally, Tunnel analysis results show that the major tunnel of Din, Flu, and Teb is located between helix K, FG loop, and β4 hairpin (Tunnel II).The top ranked possible tunnel (Tunnel II) corresponds to Vor exits through helix K, F and helix J. This study further revealed the CYP51B relevant structural characteristics at the atomic level as well as provided a basis for rational design of new and more efficacious antifungal agents.

Anthelmintic activity of cytochrome P450 inhibitors miconazole and clotrimazole: in-vitro effect on the liver fluke Opisthorchis felineus

Discovery of drugs for the treatment of opisthorchiasis and schistosomiasis is a high priority. The basic metabolic cytochrome P450 (CYP) system in parasitic flatworms contains a single gene. CYP of the liver fluke Opisthorchis felineus, the causative agent of opisthorchiasis, is important for survival of the worm, so it may be a promising target for therapeutics against liver fluke infection.The aims of this study were: (i) to analyse in-vitro anthelmintic activity of various CYP inhibitors using standard motility and mortality assays against juvenile and adult O. felineus worms; and (ii) to characterize their anthelminthic effects. Azole inhibitors (ketoconazole, miconazole, triadimenol, clotrimazole and 4-phenyl imidazole) and other inhibitors of haem-containing enzymes (disulfiram, metyrapone, benzyl isothiocyanate, and ticlopidine) were tested. This study revealed that inhibitors of haem mono-oxygenase enzymes possess anthelmintic activity. The most effective anthelmintic agents against the newly excysted metacercariae (NEM) were the antifungal agents miconazole [concentration to reduce the response by 50% (IC50) 0.79 µM] and clotrimazole (IC50 1.25 µM), both approved by the US Food and Drug Administration. The activity of miconazole and clotrimazole was comparable to that for praziquantel (IC50 0.98 µM). In addition, 100% mortality was observed among NEM after 1 d of treatment with 10 µM miconazole, after 3 d of treatment with 10 µM clotrimazole, or after 7 d of treatment with 40 µM ketoconazole. When various CYP inhibitors were tested on adult worms, clotrimazole, miconazole and ketoconazole were found to be the most effective (IC50 13–20 µM). It is speculated that CYP may represent a promising drug target for combined treatment with other anthelmintic agents. The use of inhibitor–drug combinations may improve the action of standard anthelmintic agents.

Comparison of Cytochrome P450 3A4 and 3A7 with Azole Inhibitors

Cytochrome P450 (CYP) 3A enzymes metabolize up to 50% of human drugs. While CYP3A4 is the major adult enzyme, CYP3A7 is the major form through 6–12 months of development. Though 87% identical and frequently assumed to be functionally very similar, it has recently become clear that there are some significant differences between CYP3A4 and CYP3A7 that may affect fetal and infant drug metabolism. This knowledge gap motivates a direct study of CYP3A7 metabolism and inhibition to better inform drug usage in very young patients.Azoles can be effective for common fungal infections in infants, but can also be rather nonselective CYP inhibitors. In addition to the clinical relevance of these compounds, azoles constitute a useful series of CYP3A active site probes. This study systematically evaluated the interactions of a series of azoles with purified, recombinant human CYP3A4 and CYP3A7. Binding studies revealed that each of the following azoles demonstrated spectral features typical for direct coordination to the heme iron (type II binding) with parallel rank order affinities for the two enzymes: from subnanomolar Kd values for clotrimazole, to moderate affinity for liarozole and ketoconazole, and low affinity for imidazole. In contrast, fluconazole and fosfluconazole demonstrated more unusual binding characteristics that differed between the two CYP3A enzymes. Fluconazole binding to CYP3A7 resulted in only increasing absorbance, migrating at higher concentrations to wavelengths associated with heme coordination. In contrast, fluconazole binding to CYP3A4 was characteristic of water displacement from the heme (type I) at low concentrations, but converted to heme coordination at high concentrations, suggesting that CYP3A4 may bind fluconazole in different orientations, perhaps depending on whether one fluconazole molecule or two are bound in the active site simultaneously. This would be consistent with the established capacity of CYP3A4 to bind more than one azole molecule simultaneously. Fosfluconazole, a more water‐soluble phosphorylated version of fluconazole, demonstrated a red‐shifted type I spectrum with CYP3A4, but with CYP3A7 yielded only increasing absorbance consistent with water displacement from the iron. Overall, these observations establish differences between CYP3A7 and CYP3A4 interactions with fluconazole and fosfluconazole, prompting further evaluation by comparative inhibition studies and structural studies. Such differences may underlie differential metabolism of common drugs at different life stages and inform dosing in infants vs. adults.Support or Funding InformationNational Institutes of Health R37GM076343 (EES)