Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: role of phenolic hydroxyl groups in the resorcinol moiety

AimsIn this study, we examined the inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major cannabinoids, on the activity of human cytochrome P450 (CYP) 3A enzymes. Furthermore, we investigated the kinetics and structural requirement for the inhibitory effect of CBD on the CYP3A activity.Main methodsDiltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver microsomes (HLMs) in the presence of cannabinoids was determined.Key findingsAmong the three major cannabinoids, CBD most potently inhibited CYP3A4 and CYP3A5 (IC₅₀ = 11.7 and 1.65 μM, respectively). The IC₅₀ values of Δ⁹-THC and CBN for CYP3A4 and CYP3A5 were higher than 35 μM. For CYP3A7, Δ⁹-THC, CBD, and CBN inhibited the activity to a similar extent (IC₅₀ = 23–31 μM). CBD competitively inhibited the activity of CYP3A4, CYP3A5, and HLMs (K_i = 1.00, 0.195, and 6.14 μM, respectively). On the other hand, CBD inhibited the CYP3A7 activity in a mixed manner (K_i = 12.3 μM). Olivetol partially inhibited all the CYP3A isoforms tested, whereas d-limonene showed lack of inhibition. The lesser inhibitory effects of monomethyl and dimethyl ethers of CBD indicated that the ability of CYP3A inhibition by the cannabinoid attenuated with the number of methylation on the phenolic hydroxyl groups in the resorcinol moiety.SignificanceThis study indicated that CBD most potently inhibited catalytic activity of human CYP3A enzymes, especially CYP3A4 and CYP3A5. These results suggest that two phenolic hydroxyl groups in the resorcinol moiety of CBD may play an important role in the CYP3A inhibition.     

Dual Effects of Ketoconazole cis-Enantiomers on CYP3A4 in Human Hepatocytes and HepG2 Cells

Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (−)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (−)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, K_i 0.92 µM for testosterone, IC₅₀ 1.46 µM, K_i 2.52 µM for midazolam; (−)-ketoconazole IC₅₀ 0.90 µM, K_i 0.17 µM for testosterone, IC₅₀ 1.04 µM, K_i 1.51 µM for midazolam).     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Metabolism of carbosulfan II. Human interindividual variability in its in vitro hepatic biotransformation and the identification of the cytochrome P450 isoforms involved

This study aims to characterize interindividual variability and individual CYP enzymes involved in the in vitro metabolism of the carbamate insecticide carbosulfan. Microsomes from ten human livers (HLM) were used to characterize the interindividual variability in carbosulfan activation. Altogether eight phase I metabolites were analyzed by LC–MS. The primary metabolic pathways were detoxification by the initial oxidation of sulfur to carbosulfan sulfinamide (‘sulfur oxidation pathway’) and activation via cleavage of the nitrogen sulfur bond (N–S) to give carbofuran and dibutylamine (‘carbofuran pathway’). Differences between maximum and minimum carbosulfan activation values with HLM indicated nearly 5.9-, 7.0, and 6.6-fold variability in the k_m, V_max and CL_int values, respectively. CYP3A5 and CYP2B6 had the greatest efficiency to form carbosulfan sulfinamide, while CYP3A4 and CYP3A5 were the most efficient in the generation of the carbofuran metabolic pathway. Based on average abundances of CYP enzymes in human liver, CYP3A4 contributed to 98% of carbosulfan activation, while CYP3A4 and CYP2B6 contributed 57 and 37% to detoxification, respectively. Significant correlations between carbosulfan activation and CYP marker activities were seen with CYP3A4 (omeprazole sulfoxidation), CYP2C19 (omeprazole 5-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation), displaying r² = 0.96, 0.87 and 0.82, respectively. Activation and detoxification pathways were inhibited by ketoconazole, a specific CYP3A4 inhibitor, by 90–97% and 47–94%, respectively. Carbosulfan inhibited relatively potently CYP3A4 and moderately CYP1A1/2 and CYP2C19 in pooled HLM. These results suggest that the carbosulfan activation pathway is more important than the detoxification pathway, and that carbosulfan activation is predominantly catalyzed in humans by CYP3A4.     

Design,synthesis and biological evaluation of new carbazole derivatives as anti-cancer and anti-migratory agents

Based on the efficacy of EHop-016 as an inhibitor of migration and Rac1 activation, a new series of carbazole derivatives has been synthesized. Cytotoxic and anti-migratory effects of these compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. Preliminary investigations of their anticancer activity demonstrated that several compounds have moderate antiproliferative effects on cancer cell lines with GI₅₀ values in the range of 13–50?µM. Furthermore, compounds 3b and 11b inhibit migration activity of metastatic cell line MDA-MB-231 by 32% and 34%, respectively. Compound 11b was shown to inhibit activation of the Rho GTPase Rac1 by 55% at 250?nM in both MDA-MB-231 and MDA-MB-435 cell lines. Compared with the IC₅₀ of Rac1 inhibition by lead compound EHop-016 of 1.1?µM, compound 11b demonstrates 4X improved in vitro efficacy.     

Characterization of chicken cytochrome P450 1A4 and 1A5: Inter-paralog comparisons of substrate preference and inhibitor selectivity

The chicken (Gallus gallus) is one of the most economically important domestic animals and also an avian model species. Chickens have two CYP1A genes (CYP1A4 and CYP1A5) which are orthologous to mammalian CYP1A1 and CYP1A2. Although the importance of chicken CYP1As in metabolism of endogenous compounds and xenobiotics is well recognized, their enzymatic properties, substrate preference and inhibitor selectivity remain poorly understood. In this study, functional enzymes of chicken CYP1A4 and CYP1A5 were successfully produced in Escherichia coli (E. coli). The substrate preference and inhibitor specificity of the two chicken CYP1As were compared. Kinetic results showed that the enzymatic parameters (K_m, V_max, V_max/K_m) for ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) differed between CYP1A4 and CYP1A5, while no significant difference was observed for methoxyresorufin O-demethylase (MROD). Lower K_m of CYP1A4 for BROD suggests that CYP1A4 has a greater binding affinity to benzyloxyresorufin than either ethoxyresorufin or methoxyresorufin. The highest V_max/K_m ratio was seen in BROD activity for CYP1A4 and in MROD for CYP1A5 respectively. These results indicate that substrate preference of chicken CYP1As is more notably distinguished by BROD activity and CYP1A5 prefers shorter alkoxyresorufins resembling its mammalian ortholog CYP1A2. Differential patterns of MROD inhibition were observed between CYP1As and among the five CYP inhibitors (α-naphthoflavone, furafylline, piperonyl butoxide, erythromycin and ketoconazole). α-Naphthoflavone was determined to be a potent MROD inhibitor of both CYP1A4 and CYP1A5. In contrast, no or only a trace inhibitory effect (< 15%) was observed by erythromycin at a concentration of 500 μM. Stronger inhibition of MROD activity was found in CYP1A5 than CYP1A4 by relatively small molecules α-naphthoflavone, piperonyl butoxide and furafylline. AROD kinetics and inhibition profiles between chicken CYP1A4 and CYP1A5 demonstrate that the two paralogous members of the CYP1A subfamily have distinct enzymatic properties, reflecting differences in the active site geometry between CYP1A4 and CYP1A5. These findings suggest that CYP1A4 and CYP1A5 play partially overlapping but distinctly different physiological and toxicological roles in the chicken.     

Discovery and characterization of N-(1,3-dialkyl-1H-indazol-6-yl)-1H-pyrazolo[4,3-b]pyridin-3-amine scaffold as mGlu4 positive allosteric modulators that mitigate CYP1A2 induction liability

Previous reports from our laboratory disclosed the structure and activity of a novel 1H-pyrazolo[4,3-b]pyridine-3-amine scaffold (VU8506) which showed excellent potency, selectivity and in vivo efficacy in preclinical rodent models of Parkinson’s disease. Unfortunately, this compound suffered from significant CYP1A2 induction as measured through upstream AhR activation (125-fold) and thus was precluded from further advancement in chronic studies. Herein, we report a new scaffold developed recently which was systematically studied in order to mitigate the CYP1A2 liabilities presented in the earlier scaffolds. We have identified a novel structure that maintains the potency and selectivity of other mGlu₄ PAMs, leading to 9i (hmGlu₄ EC₅₀?=?43?nM; AhR activation?=?2.3-fold).     

Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit

Background

The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas'' disease chemotherapy is sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.

Methodology/Principal Finding

In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51_Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51_Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51_Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site.

Conclusions/Significance

CYP51_Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51_Tc.     

Investigation of cytochrome P450 inhibitory properties of maslinic acid,a bioactive compound from Olea europaea L., and its structure–activity relationship

Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with IC₅₀ value at 46.1 and 62.3 µM, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with IC₅₀ values more than 100 µM. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with K_i of 18.4, 98.7 and 66.3 µM, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high K_i values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs.     

Human,rat and crocodile liver microsomal monooxygenase activities measured using diazepam and nifedipine: effects of CYP3A inhibitors and relationship to immunochemically detected CYP3A apoprotein

Nifedipine oxidase and diazepam C₃-hydroxylase were tested as activities for selectively measuring CYP3A enzymes using liver microsomes from male and female human organ donors, male and female Wistar rats and male and female estuarine crocodiles. The association between CYP3A enzymes and these monooxygenations was confirmed for the human samples. Male rat samples had lower specific contents of CYP3A apoprotein than the human samples but had equivalent (nifedipine) or higher (diazepam) monooxygenase specific activities. CYP3A apoprotein was undetectable in female rat samples which had very low activities towards both substrates. Enzyme inhibition studies showed that diazepam C₃-hydroxylase of male rat liver was attributable to CYP3A but corresponding results for female rats suggested a contribution from non-CYP3A enzyme. Western blotting with immunochemical detection using anti-CYP3A4 IgG suggested the presence of putative CYP3A apoprotein in male and female crocodile liver samples and inhibition studies with diazepam as substrate suggested the presence of CYP3A subfamily monooxygenase activity in these enzyme preparations. Results for nifedipine oxidase with male and female rat liver and male crocodile liver suggested major contributions to catalysis from non-CYP3A enzymes. Inhibition studies suggested that a higher proportion of nifedipine oxidase in female crocodile liver may be attributable to the putative CYP3A enzyme(s) than in male crocodile liver. These results show the need for care in the assessment of CYP3A activity of fractionated tissues when using these substrates in cross-species studies and where gender is a variable.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition

Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopyridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q–u); however, most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI, we introduced electron withdrawing groups on the thiophene and found that a CF₃ group on the ring or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344 rats.     

TNP and its analogs: Modulation of IP6K and CYP3A4 inhibition

Inositol hexakisphosphate kinase (IP6K) is an important mammalian enzyme involved in various biological processes such as insulin signalling and blood clotting. Recent analyses on drug metabolism and pharmacokinetic properties on TNP (N²-(m-trifluorobenzyl), N⁶-(p-nitrobenzyl)purine), a pan-IP6K inhibitor, have suggested that it may inhibit cytochrome P450 (CYP450) enzymes and induce unwanted drug-drug interactions in the liver. In this study, we confirmed that TNP inhibits CYP3A4 in type I binding mode more selectively than the other CYP450 isoforms. In an effort to find novel purine-based IP6K inhibitors with minimal CYP3A4 inhibition, we designed and synthesised 15 TNP analogs. Structure-activity relationship and biochemical studies, including ADP-Glo kinase assay and quantification of cell-based IP7 production, showed that compound 9 dramatically reduced CYP3A4 inhibition while retaining IP6K-inhibitory activity. Compound 9 can be a tool molecule for structural optimisation of purine-based IP6K inhibitors.     

Cycloartane-type triterpenes from Euphorbia fischeriana stimulate human CYP3A4 promoter activity

A chemical study of the roots of Euphorbia fischeriana resulted in the isolation of seven triterpenes (1–7), including two new compounds: (24R,S)-3β-24,31-epoxy-24-methylcycloartane (1) and (24R,S)-3β,31-dihydroxy-24-methoxy-24-methylcycloartane (2). Their structures were elucidated through extensive spectroscopic analyses. Cycloartanes 1–4 showed significant human CYP3A4 promoter activity through a series of luciferase reporter assays. Of these compounds, 3 and 4 activated the pregnane X receptor (PXR) and induced CYP3A4 mRNA expression in human primary hepatocytes. However, despite showing the most potent human CYP3A4 promoter activity via a PXR-independent pathway, 2 did not affect CYP3A4 mRNA expression in human primary hepatocytes. This difference is correlated to substitutions in C-24 and C-25 of the cycloartane structure.     

Protein-protein interactions in the systems of cytochromes P450 3A4 and 3A5

Molecular interactions between protein partners of the monooxygenase system involved in drug biotransformation (cytochromes P450 3A4, 3A5 and cytochrome b ₅) have been investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various forms of cytochrome b ₅, including microsomal (b ₅ mc) and mitochondrial (b ₅ om) forms of this protein, as well as two chimeric constructs (b ₅(om-mc),(b ₅(mc-om). Interestingly, significant differences were observed only during interactions with b ₅ om. Electroanalytical characteristics of electrodes with immobilized hemoproteins have been determined for CYP3A4, CYP3A5, b ₅ mc, b ₅ om, b ₅ mc(om-mc), and b ₅ mc(mc-om). The electrochemical analysis revealed that these proteins are characterized by close reduction potentials ranged from ?0.435 V to ?0.350 V (vs. Ag/AgCl). Cytochrome b ₅ mc stimulated the electrocatalytic activity of CYP3A4.     

Investigation of the insulin-like properties of zinc(II) complexes of 3-hydroxy-4-pyridinones: identification of a compound with glucose lowering effect in STZ-induced type I diabetic animals

Results from an investigation in an in vivo model of STZ-induced diabetic rats demonstrate that compound bis(1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate)zinc(II), Zn(dmpp)₂, significantly lowers the blood glucose levels of individuals, thus showing evidence of glucose lowering activity.The compound was selected from a set of eight zinc(II) complexes of 3-hydroxy-4-pyridinones with diverse lipophilicity that were prepared and characterized in our laboratory. Assessment of insulin-like activity of the complexes was firstly performed in vitro by measuring the inhibition of FFA release in isolated rat adipocytes. The results indicate that compounds bis(2-methyl-3-hydroxy-4-pyridinonate)zinc(II), Zn(mpp)₂ and Zn(dmpp)₂ display significantly higher activity than that of the respective positive control thus suggesting its selection for in vivo tests.Safety evaluation of the active zinc(II) compounds was performed in freshly isolated rat hepatocytes. The results support that cell viability is not significantly different from the control set after 1 and 2 h of incubation with both zinc(II) complexes.     

Discovery of quinolinone derivatives as potent FLT3 inhibitors

Recently some fms-like tyrosine kinase 3 (FLT3) inhibitors have shown good efficacy in acute myeloid leukemia (AML) patients. In an effort to develop anti-leukemic drugs, we investigated quinolinone derivatives as novel FLT3 inhibitors. Two substituted quinolinones, KR65367 and KR65370 were subjected to FLT3 kinase activity assay and showed potent inhibition against FLT3 kinase activity in vitro, with IC₅₀ of 2.7 and 0.57 nM, respectively. As a measure of selectivity, effects on the activity of other kinases were also tested. Both compounds have negligible activity against Met, Ron, epidermal growth factor receptor, Aurora A, Janus kinase 2, and insulin receptor; with IC₅₀ greater than 10 μM. KR compounds showed strong growth inhibition in MV4;11 AML cells and increased the apoptotic cell death in flow cytometric analyses. A decrease in STAT5 phosphorylation by KR compounds was observed in MV4;11 cells. Furthermore, in vitro evaluation of compounds structurally related to KR65367 and KR65370 showed a good structure-activity relationship.     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.