Fungal Cytochrome P450 Enzyme Research Articles

Oteseconazole: A Review

Many women are affected by vaginal fungal infections, also called yeast infections. Vulvovaginal candidiasis (VVC) is a common fungal infection caused by Candida species, predominantly Candida albicans. RVVC compromises with women’s life. It causes symptoms – such as itching, pain, dyspareunia, dysuria, and leucorrhea2-4. Oteseconazole – a novel, oral, selective fungal cytochrome P450 enzyme 51 inhibitor, designed to avoid off-target toxicities. In clinical studies to date, oteseconazole has demonstrated impressive efficacy, a positive tolerability profile and hope for a superior RVVC treatment option. Fungal infection comes in different form like –ringworm athelets foot, toenail fungus, yeast infection and jock itch. Yeast infection of vagina and tissue at opening of vagina. Antifungal is used to treat and prevent mycosis. In this article we studied about the new FDA approved drug for vaginal yeast infection.

Elucidation of the Complete Biosynthetic Pathway of Phomoxanthone A and Identification of a Para-Para Selective Phenol Coupling Dimerase.

Fungal cytochrome P450 enzymes have been shown to catalyze regio- and stereoselective oxidative intermolecular phenol coupling. However, an enzyme capable of catalyzing undirected para-para (C4-4') coupling has not been reported. Here, we revealed the biosynthetic gene cluster (BGC) of phomoxanthone A from the marine fungus Diaporthe sp. SYSU-MS4722. We heterologously expressed 14 biosynthetic genes in Aspergillus oryzae NSAR1 and found that PhoCDEFGHK is involved in the early stage of phomoxanthone A biosynthesis to give chrysophanol and that chrysophanol is then processed by PhoBJKLMNP to yield penexanthone B. A feeding experiment suggested that PhoO, a cytochrome P450 enzyme, catalyzed the regioselective oxidative para-para coupling of penexanthone B to give phomoxanthone A. The mechanism of PhoO represents a novel enzymatic 4,4'-linkage dimerization method for tetrahydroxanthone formations, which would facilitate biosynthetic engineering of structurally diverse 4,4'-linked dimeric tetrahydroxanthones.

Oteseconazole: an advance in treatment of recurrent vulvovaginal candidiasis.

Recurrent vulvovaginal candidiasis (RVVC) has significant disease, financial and quality-of-life burdens, affects women from all strata of society worldwide, and lacks an approved therapeutic solution. Fluconazole emerged in 2004 as an antifungal for RVVC; it provides symptom control and has been accepted worldwide as afirst-line treatment. Its limitations include thedevelopment of resistance and a high rate of vulvovaginal candidiasis recurrence after therapy cessation. There is now an improved treatment option on the horizon: oteseconazole-a novel, oral, selective fungal cytochrome P450 enzyme 51 inhibitor, designed to avoid off-target toxicities. In clinical studies to date, oteseconazole has demonstrated impressive efficacy, a positive tolerability profile and hope for a superior RVVC treatment option.

Sterol 14α-Demethylase Ligand-Binding Pocket-Mediated Acquired and Intrinsic Azole Resistance in Fungal Pathogens.

The fungal cytochrome P450 enzyme sterol 14α-demethylase (SDM) is a key enzyme in the ergosterol biosynthesis pathway. The binding of azoles to the active site of SDM results in a depletion of ergosterol, the accumulation of toxic intermediates and growth inhibition. The prevalence of azole-resistant strains and fungi is increasing in both agriculture and medicine. This can lead to major yield loss during food production and therapeutic failure in medical settings. Diverse mechanisms are responsible for azole resistance. They include amino acid (AA) substitutions in SDM and overexpression of SDM and/or efflux pumps. This review considers AA affecting the ligand-binding pocket of SDMs with a primary focus on substitutions that affect interactions between the active site and the substrate and inhibitory ligands. Some of these interactions are particularly important for the binding of short-tailed azoles (e.g., voriconazole). We highlight the occurrence throughout the fungal kingdom of some key AA substitutions. Elucidation of the role of these AAs and their substitutions may assist drug design in overcoming some common forms of innate and acquired azole resistance.

Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system.

The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11β-/14α-hydroxylase P-450lun from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11β-hydroxylation activity of the P-450lun towards cortexolone (RSS) substrate; however, the potential steroid 14α-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14α-hydroxylation activity of the P-450lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450lun functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450lun-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450lun and CPRlun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.

Biodegradation and bioconversion of endrin by white rot fungi, Phlebia acanthocystis and Phlebia brevispora

Endrin is persistent organic pollutants that contaminate soil in many parts of the world. In this study, endrin was used as the substrate for a degradation experiment with the white rot fungi of the genus Phlebia . The results of tolerance test showed that the tolerance level of Phlebia acanthocystis and Phlebia brevispora to endrin was higher than that of other fungi, and the tolerance coefficient of both strains to 1.0 mg/L endrin exceeded 0.9 in solid PDA medium. P. acanthocystis and P. brevispora could degrade endrin efficiently in pure culture, especially P. acanthocystis had the highest degradability of more than 80% after 20 d incubation. Compared with low-nitrogen medium, PDB medium is more suitable for the biodegradability of two fungi. Several hydroxylated products such as 8-hydroxyendrin and two monohydroxyendrin were detected, indicating that endrin was initially branched to different monohydroxylated products in fungal degradation. Moreover, a carboxylic acid product was obtained from P. acanthocystis culture, suggesting that the carboxylation reaction occurred in bioconversion of endrin. The fungal cytochrome P450 enzymes play significant role in the in the initial hydroxylation process on endrin degradation. This is the first report that endrin is converted to hydroxylated and carboxylated metabolites by microorganisms. • Phlebia acanthocystis and P. brevispora can tolerate endrin of different concentrations. • Both strains showed excellent ability to degrade endrin in PDB and HCLN media. • Endrin was converted into several hydroxylated and carboxylated products. • Fungal Cyt P450 is involved in the initial hydroxylation of endrin.

Fluconazole, a Fungal Cytochrome P450 Enzyme Inhibitor: The Potential Role in Augmenting Hepatotoxicity and Hyperinsulinemia Induced by Dexamethasone in Rats

Insulin resistance (IR) is a widespread health problem characterized by failure of cell responses to insulin. IR increases the risk of fungal infections. Long-term administration of dexamethasone is a therapeutic need accompanied IR and fungal infections. Fluconazole is the first-line antifungal therapy that inhibit fungal cytochrome P450 (CYP450) enzyme. It has a hepatotoxic effect and moderate inhibitory effect on multiple CYP450 enzymes that could affect the pharmacokinetics of drugs used to treat insulin resistance such as metformin. This study aims to evaluate the effect of administration of fluconazole on liver function, CYP450 concentration and level of insulin resistance induced by dexamethasone and high fat diet. Insulin resistance in rats was induced by dexamethasone (0.1mg/kg/day s.c) (DEX) for 2 weeks, after eight weeks of high fat diet (HFD) until the end of the experiment. Oral administration of fluconazole (20, 40 mg/kg) (FLU20, 40) was started concomitantly with DEX for 2 weeks. Metformin (400mg/kg) (MET) was used as a reference treatment for IR and administered orally for 2 weeks with DEX and FLU (20, 40). Concomitant administration of FLU either 20mg/kg or 40 mg/kg with DEX showed significant increase in IR, AST and ALT compared by that caused by DEX alone or with MET. FLU showed a significant decrease in serum CYP450 that elevated by DEX. Massive fibrosis was noticed in histological assessments of liver tissues of HFD+DEX+FLU (20, 40) groups. In conclusion administration of FLU in case of IR induced by HFD+DEX resulted in deterioration of liver functions and decrease in the therapeutic efficacy of MET.

Cytochrome P450-catalyzed regio- and stereoselective phenol coupling of fungal natural products.

For almost 100 years, phenoxy radical coupling has been known to proceed in nature. Because of the linkage of their molecular halves (regiochemistry) and the configuration of the biaryl axis (stereochemistry), biaryls are notoriously difficult to synthesize. Whereas the intramolecular enzymatic coupling has been elucidated in detail for several examples, the bimolecular intermolecular coupling could not be assigned to one single enzyme in the biosynthesis of axially chiral biaryls. As these transformations often take place regio- and stereoselectively, enzyme-catalyzed control is reasonable. We now report the identification and expression of fungal cytochrome P450 enzymes that catalyze regio- and stereoselective intermolecular phenol couplings. The cytochrome P450 enzyme KtnC from the kotanin biosynthetic pathway of Aspergillus niger was expressed in Saccharomyces cerevisiae. The recombinant cells catalyzed the coupling of the monomeric coumarin 7-demethylsiderin both regio- and stereoselectively to the 8,8'-dimer P-orlandin, a precursor of kotanin. The sequence information obtained from the kotanin biosynthetic gene cluster was used to identify in silico a similar gene cluster in the genome of Emericella desertorum, a producer of desertorin A, the 6,8'-regioisomer of orlandin. The cytochrome P450 enzyme DesC was also expressed in S. cerevisiae and was found to regio- and stereoselectively catalyze the coupling of 7-demethylsiderin to M-desertorin A. Our results show that fungi use highly specific cytochrome P450 enzymes for regio- and stereoselective phenol coupling. The enzymatic activities of KtnC and DesC are relevant for an understanding of the mechanism of this important biosynthetic step. These results suggest that bimolecular phenoxy radical couplings in nature can be catalyzed by phenol-coupling P450 heme enzymes, which might also apply to the plant kingdom.

Possible association between fluconazole administration and acute hyperkalemia in a critically ill cyanotic infant.

Fluconazole, an azole antifungal agent, inhibits the fungal cytochrome P450 enzyme. Although in vitro data show that fluconazole is not a potent inhibitor of mammalian P450 enzymes, hyperkalemia secondary to adrenal insufficiency associated with fluconazole has been described rarely [1–3]. However, life-threatening severe hyperkalemia related to administration of intravenous fluconazole without adrenal suppression has not been reported before. A 2.5-month-old male infant diagnosed with tetralogy of Fallot with pulmonary atresia and multiple aorto-pulmonary collateral vessels was transferred to a pediatric intensive care unit due to septicemia. On physical examination, he was tachypneic and tachycardic with decreased oxygen saturation (62%) and a temperature of 38.8°C. Laboratory studies included hemoglobin, 12.4 g/dl; white blood count, 23.2 × 109/l; C-reactive protein, 38.3 mg/l (0–3.2); and serum potassium, 3.8 mEq/l (3.5–5.1 mEq/l). Other biochemical parameters and renal function tests were in the normal range. Blood cultures were positive for coagulase negative Staphylococcus. He was started on teicoplanin and meropenem intravenously. As fever persisted after 48 h with new infiltrates on chest radiography, and tracheal colonization with Candida spp., fluconazole 5 mg/kg intravenously once a day was given. After 24 h of treatment with fluconazole, bradycardia developed in the patient. Electrocardiography showed a prolonged PR interval and widening of the QRS complex with bradycardia (Figure 1). His simultaneous laboratory testing demonstrated a serum potassium level of 8 mEq/l. He was treated with repetitive administration of sodium bicarbonate, insulin/glucose, as well as calcium gluconate. Repeat blood results showed potassium 11 mEq/l. The results of renal and liver function tests and the levels of other electrolytes were within normal limits. There were no signs of rhabdomyolysis, hemolysis or severe acidosis. No medication errors could be identified. Infusion of fluconazole was stopped, and he was treated with repetitive administration of bicarbonate, insulin/glucose, and albuterol by nebulizer. In the meantime peritoneal dialysis was administered in the patient. The serum potassium level gradually returned to a normal level (4 mEq/l), and the findings of ECG abnormalities were improved. Figure 1 ECG shows widened QRS and prolonged PR interval Prophylaxis with fluconazole has been shown to reduce the occurrence of candidiasis in intensive care units [4, 5]. Even though fluconazole in standard doses is thought to be safe, there are rare reports of adrenal suppression associated with fluconazole administration [2, 3]. In our patient, severe hyperkalemia was observed after 24 h of treatment with fluconazole. Our patient's renal functions remained normal and evidently did not impair potassium excretion. No evidence of rhabdomyolysis, severe acidosis, hemolysis or adrenal suppression was detected. Although the causative mechanism is unclear, severe hyperkalemia was associated with fluconazole in our patient. All other possible causes of hyperkalemia were excluded. Fluconazole may have unknown life-threatening side-effects, despite having been tested prior to its clinical use. To our knowledge, this is the first report of a life-threatening complication of fluconazole without adrenal insufficiency. We speculate that because of chronic hypoxia of our patient due to his cyanotic heart disease, the drug side effect may have been triggered, and it may be prudent to monitor electrolytes especially in critically ill cyanotic patients.

Functional Expression and Characterization of Recombinant NADPHP450 Reductase from Malassezia globosa

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

Biotransformation of the organochlorine pesticide trans-chlordane by wood-rot fungi

There is very limited information on the biotransformation of organochlorine pesticide chlordane by microorganisms, and no systematic study on the metabolic products and pathways for chlordane transformation by wood-rot fungi has been conducted. In this study, trans-chlordane was metabolized with the wood-rot fungi species Phlebia lindtneri, Phlebia brevispora and Phlebia aurea, which are capable of degrading polychlorinated dibenzo-p-dioxin and heptachlor epoxide. At the end of 42 days of incubation, over 50% of trans-chlordane was degraded by the fungal treatments in pure cultures. These fungi transformed trans-chlordane to at least eleven metabolites including a large amount of hydroxylated products such as 3-hydroxychlordane, chlordene chlorohydrin, heptachlor diol, monohydroxychlordene and dihydroxychlordene. P. lindtneri particularly can metabolize oxychlordane, a recalcitrant epoxide product of chlordane, into a hydroxylated product through substitution of chlorine atom by hydroxyl group. The present results suggest that hydroxylation reactions play an important role in the metabolism of trans-chlordane by these Phlebia species. Additionally, transformation of trans-chlordane and production of hydroxylated metabolites were efficiently inhibited by the addition of cytochrome P450 inhibitors, piperonyl butoxide and 1-aminobenzotriazole, demonstrating that fungal cytochrome P450 enzymes are involved in some steps of trans-chlordane metabolism, particularly in the hydroxylation process.

Synthesis and In Vitro Evaluation of Novel 1, 2, 4-Triazole Derivatives as Antifungal Agents

Despite the advances in medicine and the emergence of new antifungal agents, fungal infections remain a significant cause of morbidity and mortality. Azoles are widely used as antifungal agents. Azoles interfere with the conversion of lanosterol to ergosterol by inhibiting a fungal cytochrome P450enzyme, lanosterol 14α-demethylase. Resistance to azoles, particularly fluconazole, is emerging to Candida albicans, after long-term suppressive therapy. Thus, there is an urgent need for newer potent antifungals to combat resistance developed against widely used azoles. In present work, we report synthesis of novel triazole derivatives of 7-hydroxy-4-methylcoumarin using various substituted aromatic aldehydes and evaluated for their in vitro fungicidal activity against Candida albicans at various concentrations to obtain minimum inhibitory concentration (MIC).

Prednisone reduces ketoconazole-induced skeletal defects in rat fetuses

Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not specific, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also affected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic effects remains to be elucidated. When considering the potential inhibitory effect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis.

Photoaging and phototoxicity from long-term voriconazole treatment in a 15-year-old girl

Voriconazole is a second-generation triazole that was approved by the Food and Drug Administration in May 2002 for treatment of severe fungal infections. In clinical trials it demonstrated superior efficacy in addition to a survival benefit when compared with the then current treatment standard, amphotericin B, for primary treatment of invasive aspergillosis. Voriconazole is a highly selective inhibitor of fungal cytochrome P450 enzymes. Adverse cutaneous reactions have been reported, namely cheilitis, erythema, discoid lupus erythematosus, Stevens-Johnson syndrome, toxic epidermal necrolysis, erythema multiforme, and photosensitivity reactions. We report a case of photoaging caused by voriconazole therapy. A 15-year-old patient developed cheilitis and erythema over the sun-exposed areas of her body 5 weeks after beginning voriconazole for a severe fungal infection. The lesions showed a mild transient improvement before subsequent photodamage occurred to the back of her forearms, back of her hands, and face. Voriconazole was discontinued once the fungal infection had completely resolved. The patient's blisters, erythema, and cheilitis resolved after discontinuation of voriconazole. However, she was left with solar elastotic changes, multiple lentigines, and ephelides of sun-exposed areas. These cutaneous manifestations may represent a unique adverse event caused by a new second-generation triazole.

Clinical impact of drug-drug interactions with systemic azole antifungals.

The number of drug-drug interactions is remarkably high among hospitalized patients receiving systemic azole antifungal agents. Recent estimates suggest that as many as 95% of hospitalized patients treated with azole antifungals may receive medications capable of producing a major or moderate pharmacokinetic interaction. The antifungal properties of the azoles stem from their propensity to inhibit fungal cytochrome P-450 enzymes. In humans, however, azole antifungals also interfere with several hepatic and intestinal cytochrome P-450 isoenzymes responsible for the metabolism of numerous drugs. As a result, the azole antifungals have drug-drug interactions with a plethora of drug classes, including H(1)-antihistamines, antineoplastics, steroids, antimicrobials, antiretrovirals, opioids, long acting barbiturates, cardiovascular agents, psychotropics and oral contraceptives. These interactions are so numerous that it is extremely difficult to remember them all and would be even harder to prospectively predict their consequences in an individual patient. In fact, any drug that shares the same cytochrome P-450 isoenzymes for metabolism may potentially to give rise to drug-drug interactions in vivo. Patients with specific polymorphisms are probably at especially high risk. Certain drug combinations with azoles should be absolutely avoided, while other combinations may be prescribed provided monitoring of drug levels is undertaken, dosage reduction of one or more of the drugs is made (as appropriate) and/or careful monitoring of clinical parameters is performed.

Plant Sterol 14α-Demethylase Affinity for Azole Fungicides

Azole fungicides were thought to have much greater affinity for the fungal cytochrome P450 enzyme, sterol 14α-demthylase (CYP51) than the plant orthologue. Using purified CYP51 from the plant Sorghum bicolor L Moenech, a direct comparison of the sensitivity to the fungicides triadimenol and tebuconazole has been carried out. S. bicolor CYP51 was purified to homogenity as determined by SDS–PAGE and specific heme content. Addition of the azole fungicides triadimenol and tebuconazole induced type II spectral changes, with saturation occurring at equimolar azole/P450 concentrations. Inhibition of reconstituted activities revealed only a threefold insensitivity of the plant CYP51 compared to a fungal CYP51, from the phytopathogen Ustilago maydis, as judged by IC50 values. The implications for fungicide mode of action and application are discussed.